Physiol Rev 94: 951–986, 2014
doi:10.1152/physrev.00038.2013
THE PHYSIOLOGY OF MECHANOELECTRICAL
TRANSDUCTION CHANNELS IN HEARING
Robert Fettiplace and Kyunghee X. Kim
Department of Neuroscience, University of Wisconsin-Madison, Madison, Wisconsin
Fettiplace R, Kim KX. The Physiology of Mechanoelectrical Transduction Channels in
Hearing. Physiol Rev 94: 951–986, 2014; doi:10.1152/physrev.00038.2013.—
Much is known about the mechanotransducer (MT) channels mediating transduction in
hair cells of the vertrbrate inner ear. With the use of isolated preparations, it is
experimentally feasible to deliver precise mechanical stimuli to individual cells and
record the ensuing transducer currents. This approach has shown that small (1–100 nm) deflections of the hair-cell stereociliary bundle are transmitted via interciliary tip links to open MT channels
at the tops of the stereocilia. These channels are cation-permeable with a high selectivity for Ca2⫹;
two channels are thought to be localized at the lower end of the tip link, each with a large
single-channel conductance that increases from the low- to high-frequency end of the cochlea. Ca2⫹
influx through open channels regulates their resting open probability, which may contribute to
setting the hair cell resting potential in vivo. Ca2⫹ also controls transducer fast adaptation and
force generation by the hair bundle, the two coupled processes increasing in speed from cochlear
apex to base. The molecular intricacy of the stereocilary bundle and the transduction apparatus is
reflected by the large number of single-gene mutations that are linked to sensorineural deafness,
especially those in Usher syndrome. Studies of such mutants have led to the discovery of many of
the molecules of the transduction complex, including the tip link and its attachments to the
stereociliary core. However, the MT channel protein is still not firmly identified, nor is it known
whether the channel is activated by force delivered through accessory proteins or by deformation
of the lipid bilayer.
L
I.
II.
III.
IV.
V.
VI.
VII.
INTRODUCTION
THE HAIR CELL AND ITS...
MECHANOTRANSDUCER CURRENTS
REVERSE TRANSDUCTION
THE MECHANOTRANSDUCER CHANNEL...
THE MOLECULES OF THE...
CONCLUSIONS
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I. INTRODUCTION
Ion channels are proteinaceous pores that regulate the flow
of cations or anions across the plasma membrane, the resulting current flow initiating the electrical signals that are
the hallmark of neural activity in animals. Common examples include the voltage-gated Na⫹ and K⫹ channels, underlying the nerve action potential, and ligand-gated channels,
such as the glutamate and GABA receptors, which mediate
excitatory and inhibitory synaptic transmission. Both types
of ion channel have been extensively characterized at a biophysical and molecular level over the last 20 years (98).
They are typically composed of four to five membranespanning protein subunits clustered around a central aqueous conduit that can be opened or closed by the appropriate
electrical or chemical stimulus. The basis of the stimulus
specificity resides within the primary subunits, either as a
voltage-sensing or a ligand-binding domain. A third more
heterogeneous and less well understood variety of ion channel comprises the mechanically sensitive channels that detect or transduce mechanical forces occurring during cellular deformation and motion. These belong to an ancient
class of channels, examples of which have been documented
in bacteria and fungi (141) as well as in invertebrate and
vertebrate animals (6). They may have first evolved as a
sensor of membrane stretch during osmotic shock as exemplified by the bacterial MscL channel or vacuolar transient
receptor potential TRP-Y1 channel (141). However, in animals ranging from nematode worms to mice, mechanosensitive channels unrelated to MscL are present in sensory
cells specialized to detect stretching of the skin or distention
of internal tissues such as the vasculature, muscle fibers, or
membranes of the inner ear. Two aspects of specialization
can be noted. The channels may be part of a multimolecular
complex that includes accessory proteins in the internal
cytoskeleton and in the extracellular matrix, the role of
which may be to focus force on the ion channel to ensure
rapid activation. The prime example is the array of MEC
proteins named from touch-insensitive mutations in the
worm Caenorhabditis elegans. Multiple accessory proteins,
including MEC-2 and MEC-6, are linked directly or indirectly to an ion channel fashioned from two pore-forming
subunits, MEC-4 and MEC-10 (6, 7). The transduction apparatus in C. elegans touch neurons is currently the most com-
0031-9333/14 Copyright © 2014 the American Physiological Society
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
pletely described of all animal mechanoreceptors. Second, the
extracellular matrix may be elaborated by addition of supporting structures to direct the mechanical stimulus to the
receptor cells, and to provide the equivalent of an impedance
matching device, to adjust the magnitude of the incident forces
to those required to operate the ion channel itself. The best
examples are the mechanoreceptors in the vertebrate skin,
where some afferent terminals are cloaked in nonneuronal
structures forming specialized endings such as Pacinian corpuscles or Merkel disks, each being designed to optimize the
afferent response for a tactile stimulus of given size and speed
(252). Thus the Merkel disks respond best to textured stimuli
slowly grazing the skin, whereas the onion-like capsules of
Pacinian corpuscles transmit only transient stimuli (170) and
are tuned to vibrations of several hundred Herz.
Nowhere is the structural refinement more evident than in
auditory receptors, for which an essential prerequisite is fast
transduction to encode high-frequency sound vibrations. A
striking case is Johnston’s chordotonal organ in Drosophila
in which the auditory neuron projects a distal sensory cilium (thought to be the site of transduction) that is inserted
into the antennal joint and ensheathed by specialized supporting cells. In the vertebrate inner ear, the sensory hair
cells also possess an external projection of 50 or more interconnected microvilli, the hair bundle, the structure of
which varies according to the location in the organ (72, 52).
In the auditory division of the inner ear, known as the
cochlea, motion of the hair bundles is driven by attachment
to an acellular matrix, the tectorial membrane (FIGURE 1);
in the vestibular division, including the saccule and utricle,
force is applied to the bundle via an otolithic membrane
that is mass loaded with calcium carbonate crystals. Auditory hair cells are probably the fastest and most sensitive of
all vertebrate mechanoreceptors, and those in the mammalian cochlea may respond to vibrations (elicited by sound
stimuli) with amplitudes of 1 nm and in some species, such
as mice or bats, at frequencies as high as 100 kHz. Adaptation to these exacting stimuli is likely to involve the accessory apparatus, the external and internal attachments of the
mechanotransducer (MT) channel within each stereocilium, and the nature of the MT channel protein itself.
Among the known mechanoreceptors, the need to fine-tune
channel activation to the attributes of the mechanical stimulus to be detected may go some way to explaining the
diversity of channel proteins employed. These range from
the pentameric MscL channel, which forms large 3-nmwide pores in E. coli membrane; the trimeric MEC channels
in C. elegans, closely related to the vertebrate epithelial Na⫹
channel (ENaC), which may be reused as ASIC1 channels in
some vertebrate cutaneous afferents; and the recently discovered piezo2, an enormous, 1.2-MDa protein tetramer
that may too be employed in fast-adapting mammalian cutaneous endings (43, 44), including the Merkel disks (268).
The molecular identity of the hair cell MT channel is still
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not firmly established. The heterogeneity of the mechanosensitive channels across taxa has undermined attempts to
isolate the hair cell MT channel by homology with proteins
of equivalent function from other taxa, a technique that
proved successful in cloning voltage-gated K⫹ channels
(98). Thus ion channels thought to be involved in transduction in Johnston’s organ are transient receptor potential
channels, NOMPC (TRPN) and NANCHUNG and INACTIVE (both TRPV channels) (6), none of which is directly
implicated in hair cell transduction (36). The most powerful
approach for identifying mechanosensitive channels in all
animals has been by studying specific mutants lacking touch
or auditory sensation; it is expected that this approach will
ultimately uncover the hair-cell MT channel.
Despite incomplete information on the makeup of the haircell transduction complex, the physiological setting and
biophysical properties of the MT channel have been better
characterized than for all other vertebrate mechanoreceptors. The accessory apparatus, organization of the stereociliary bundle, and mechanism of channel activation are
addressed in the first two sections, and the physiological
implications and biophysical properties are in the next two
sections. Available evidence on the molecular under pinning
is reviewed in the last section. Many of the seminal conclusions have stemmed from study of frog saccular hair cells
(41, 107, 109, 228), and these are described where relevant.
However, the review will focus primarily on auditory hair
cells, since their physiological context in responding to
sounds of different frequencies is best understood, and the
molecular foundations have been illuminated by studying
genetic deafness in mice and humans.
II. THE HAIR CELL AND ITS
ENVIRONMENT
A. Organization of the Cochlea
The auditory epithelium of the vertebrate inner ear, homologous in reptiles, birds, and mammals (160), consists of hair
cells firmly anchored to nonsensory supporting cells and
sandwiched between a fibrous basilar membrane and an
acellular tectorial membrane (FIGURE 1). The submicron
relative motion of these two structures elicited by sound
stimuli is detected by the hair cells. It is important to consider at the outset the functional specializations of the epithelium because these influence the mechanotransduction
process. First, it is a tight epithelium separating fluid compartments of different composition. Perilymph bathing the
basolateral aspect of the hair cell in adult rats contains 138
mM Na⫹, 7 mM K⫹, and 0.7 mM Ca2⫹ (25, 26), which,
with adequate Ca2⫹ and Na⫹, is conducive to synaptic
transmission from the hair cell and for action potential
generation in the afferent dendrites. In contrast, the top
surface of the hair cell and the hair bundle are immersed in
endolymph, which in rats has a composition of 154 mM
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HAIR CELL MECHANOTRANSDUCER CHANNELS
D
A
Tectorial membrane
0.2
0.4
0.1
0.6
0.03
Turtle
Hair bundles
Hair cells
0.2 mm
Basilar membrane
E
B
1k
Tectorial membrane
Tall hair cells
Hair bundles
0.3
2k
Short
hair cells
0.1
Chicken
4k
1 mm
Basilar membrane
F
C
Tectorial membrane
Hair bundles
30k
Outer
hair cells
5k
60k
Rat
1k
10k
Inner
hair cell
3k
15k
Basilar membrane
1 mm
Efferent
Afferents
FIGURE 1. The auditory organ in three vertebrate classes. The spatial map of best or characteristic
frequencies (CF) of neurons along the basilar membrane, often referred to as the tonotopic map, is illustrated
for members of three vertebrate classes: A, basilar papilla of the red-eared turtle, Trachmemys scripta
elegans; B, basilar papilla of the chicken, Gallus gallus domesticus; C, cochlea of the rat, Rattus norvegicus.
The CFs are expressed in kHz, with the frequency range being ⬃30 – 600 Hz in turtle (47); 100 Hz to 4 kHz
in chicken (33, 118), and 1– 60 kHz in rat (175). Transverse sections across the hair cell epithelium are
schematized on the right. D: turtle papilla contains a single type of hair cell innervated by both afferent (red) and
efferent (blue) nerve fibers. E: chicken papilla contains two extremes of hair cell: on the neural aspect tall hair
cells innervated mainly by afferents and on the abneural aspect short hair cells innervated predominantly by
efferents. Although not shown, in both turtle and chicken, each afferent collects from only one or two hair cells.
F: rat cochlea, similar to other mammals, contains three rows of outer hair cells with efferent innervations, and
one row of inner hair cells innervated by 95% of the auditory afferents.
K⫹, 1 mM Na⫹, 0.03 mM Ca2⫹, and 0.01 mM Mg2⫹ (25,
26); the low concentrations of Ca2⫹ and Mg2⫹ enhance
operation of mechanotransduction since at high levels these
two divalent ions block the MT channels (42, 46, 207). In
addition, in mammals there is up to a 100 mV positive
potential (rat, 25; mouse, 235), the endolymphatic potential, between the endolymph and perilymph, which is absent
in nonmammals. Second, with the evolution of the inner ear
from early reptiles to mammals, the epithelium has been
progressively elaborated to include more than one type of
hair cell accompanied by diversification of supporting cells,
changes associated with extension of the upper limit of the
frequency range (160). Thus, in reptiles like the turtle, there
is a single morphological class of hair cell having common
afferent and efferent innervation. At the other extreme, placental mammals possess two distinct categories of hair cell:
a single row of flask-shaped inner hair cells (IHCs), contacting the majority of the afferent fibers, and three to four rows
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
of columnar outer hair cells (OHCs); the latter play a role in
amplifying the mechanical stimulus (see sect. IVB) and have
a sparse afferent but strong efferent innervation. This division of labor is also seen in egg-laying mammals, the
monotremes, and in birds and crocodilians (160, 259), although more rows of each cell type are present and the
dichotomy between these two categories is less marked than
in mammals (246). A third feature of the auditory endorgan, known as the cochlea in mammals, is its ability to
separate sound frequencies along its length; as a consequence, nerve fibers emanating from different regions are
tuned to different frequencies, high-pitched tones at the
proximal end of the organ nearest the saccule, and low
frequencies at the distal end (67; FIGURE 1). The mapping of
frequency onto longitudinal position is referred to as the
tonotopic organization and is reflected in gradients in numerous cellular properties, including epithelial and cellular
dimensions and the ion channel complement of the hair
cells. Of particular note here is the variation in hair bundle
structure and MT channel properties depending on the animal or frequency range detected.
B. The Stereociliary Bundle
The stereociliary (hair) bundle, projecting from the top face
of the hair cell, is the organelle through which all mechanical stimuli are focused for detection by the transduction
machinery. This sensory specialization is composed of a
cohort of modified microvilli known as stereocilia, arranged in a staircase, with successive ranks increasing in
height across the bundle (FIGURE 2; Ref. 72). Abutting the
tallest rank is a microtubule-containing cilium, the kinocilium, which is present in vestibular hair cells and immature auditory hair cells, but not in the adult mammalian
cochlea (72, 196). The shaft of each component stereocilium is buttressed by a core of actin filaments that are crossbridged by a number of proteins including plastin-1 and
fascin-2 (the two most abundant; Ref. 226) and espin (277),
and extend from its very top to the narrowed ankle region;
only a fraction of the filaments traverses the ankles into the
cell body (68, 249), rendering the ankles the site of least
stiffness. Here, the actin filaments are packed more densely
than in the stereociliary shaft and are interconnected by the
actin bundling protein TRIOBP, which probably increases
the strength and durability of this region (134). With application of force to the tip of the bundle, the stereocilia bend
at their ankles and, because of the extracellular links connecting adjacent ranks, the entire array bends as one (45,
135). Transduction is polarized so that deflection of the
bundle towards its tallest edge opens MT channels, motion
in the opposite direction closes these channels, and orthogonal displacements are relatively ineffective (228). A variety
of interciliary links endows the bundle with cohesion (91),
the most important in the adult being the horizontal top
connectors in the contact region between the top of one
stereocilium and its taller neighbor (260), and the tip links
(FIGURE 3A). The top connectors, of which stereocilin may
be a component, appear during maturation of mouse cochlear hair cells between postnatal days 9 and 14, around
the onset of hearing (260). The top connectors replace other
interciliary links including transient lateral links and ankle
links (84). In stereocilin-null mice, the top connectors do
not form, leading to loss of bundle cohesion after postnatal
day 15, and the concomitant disappearance of the tip links
(260). In intact wild-type bundles, displacements of the stereocilia are believed to be transmitted to the MT channels
by a change in tension in the tip links (71, 193), which
stretch from the top of one stereocilium to the side wall of
FIGURE 2. Structure of the stereociliary bundle.
A: scanning electron micrograph of the hair bundle from a
turtle auditory hair cell exhibiting nine rows of stereocilia of
incremental heights and a kinocilium, arrowed (90). B: rat
OHC showing only three rows of stereocilia. C: rat IHC
illustrating the tall first row of stereocilia that can behave
like a “flag” to fluid flow in the subtectorial space and the
shorter and more numerous second, third, and fourth
rows (18). D: bat inner hair cell from the high-frequency
region of the cochlea showing only two rows of stereocilia.
Scale bar ⫽ 1 ␮m for A and 2 ␮m for B–D. (Micrographs
in A, B, and D courtesy of C. M. Hackney and D. N.
Furness.)
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HAIR CELL MECHANOTRANSDUCER CHANNELS
A
B
Upper TL
density
C
UTLD
Tip link
Tip link
MT channel
Lower TL
density
LTLD
MT channel
Adaptation
spring
Contact
region
0.2 µm
FIGURE 3. The tip link and connections. A: transmission electron micrograph of a guinea pig outer hair cell
illustrating the tip link and the electron-dense regions at the upper and lower ends, denoted as the upper tip-link
density (UTLD) and lower tip-link density (LTLD), respectively. Note the membrane has pulled away from the
LTLD where it attaches to the tip link, and there is an electron-dense “contact region” where the shorter
stetereocilium abuts the taller one. This may be the site of the top connectors. Scale bar ⫽ 0.2 ␮m. B: one
theoretical scheme for the connections of the MT channel, where two channels float free in the plasma
membrane. C: another scheme where each MT channel is attached to one strand of the tip link and is also
anchored to the internal cytoskeleton by an “adaptation spring.” Two intermediate arrangements have been
proposed in which the MT channels are connected only to the tip link or only to the internal cytoskeleton (194).
its taller neighbor. Various models have been suggested to
explain transmission of force from the tip links to the channels (e.g., Ref. 194). At one extreme, the channels float free
in the plasma membrane and are activated by a change in
membrane tension (FIGURE 3B); at the other extreme, the
channels are immobilized by connections to the tip link
externally and the cytoskeleton internally (FIGURE 3C), and
act as force sensors between these two attachments. There
are intermediate models in which the channels are connected only to the tip links or only to the cytoskeleton.
The tip links are helical extracellular filaments ⬃150 nm in
length and, in transmission electron micrographs, the end
points of attachment to the stereocilia are marked by electron-dense regions referred to as the upper tip-link density
(UTLD) and lower tip-link density (LTLD) (73, 121). Detailed evidence for the role of the tip links and the MT
channel localization at the lower end of the tip links (20)
will be described in section VB. The directionality of the
transduction mechanism can be largely accounted for by the
uniform orientation of the tip links which to a first approximation run parallel to the bundle’s plane of symmetry
(193). The directionality conforms to a cosine rule (228),
the probability of MT channel opening being proportional
to cos␪, where ␪ is the angle between the direction of the
stimulus and the bundle’s axis of symmetry. For perspective, if ␪ ⫽ 45°, cos␪ ⫽ 0.71, so the directionality is not
highly tuned, and there is some variance in the polarity of
the tip links further weakening the directional tuning. Nevertheless, in the adult cochlea the hair bundles, all have a
common orientation. The “V”-shaped bundles point away
from the neural limb, so the hair cells are maximally excited
by upward motion of the cochlear partition towards the
scala media, producing radial deflection of the tectorial
membrane and hair bundles. The uniform bundle orientation and the molecular events that establish it during development are collectively referred to as “planar cell polarity”
and involve multiple signaling pathways (169). The directionality to transduction is a feature of mature hair bundles,
and there are exceptions to this rule in immature mammals
(265). Another important property that emerges from the
structure of the bundle is that it behaves as a transformer.
For small stimuli within the physiological range, displacements occurring at the channel are reduced relative to those
at the bundle’s tip by a geometrical factor ␥ (77, 166, 192),
given approximately by the ratio of s, the distance along the
axis of symmetry between the pivot points at the stereociliary ankles (⬍1 ␮m) to the mean stereociliary height h (1–10
␮m); ␥ depends on the bundle dimensions and in most cases
lies between 0.1 and 0.5 (166). Detailed analysis of several
hair bundle morphologies, ranging from those with many
rows, as in the bullfrog saccule, to these with only two or
three rows, such as guinea pig and bat IHCs (see FIGURE 2),
showed that the gain ␥ was approximately constant across
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
the bundle if the interciliary coupling was tight. Furthermore, despite differences in the step height from one row to
the next, ␥ is largely determined by the separation of adjacent rootlets and the overall height of the bundle (192).
The dimensions and organization of the hair bundle vary
considerably depending on the end organ and the frequency
range detected. The height of the tallest rank extends from
⬃1 to 10 ␮m, and the number of stereocilia ranges from a
few dozen to a few hundred. In vestibular and nonmammalian auditory hair cells, there may be up to 10 ranks of
stereocilia across the bundle (FIGURE 2A), but in the mammalian cochlea, only two to three rows are present (FIGURE
2, B–D). In auditory hair cells there are also variations along
the tonotopic axis (turtle, Ref. 90; rat, Ref. 216; chicken,
Ref. 248). In the chicken auditory papilla, the number of
stereocilia per bundle increases and the bundle height decreases, both by a factor of five, in progressing from the lowfrequency to high-frequency end of the papilla (248); together
these changes make the bundle stiffness vary 100-fold from
one end of the papilla to the other (sect. IVB). Similar though
smaller variations are seen in mammalian cochlear OHCs,
where maximum bundle height decreases from 6 to 1.5 ␮m
and the number of stereocilia increases from ⬃60 to 100 (18,
153, 216). As discussed later (see sect. IVB), hair bundle mechanical stiffness is inversely proportional to the square of the
bundle height, so changes in height occurring in the cochlea
can have a large effect on bundle stiffness.
The diversity in bundle morphology will have a number of
outcomes for the response properties of the bundles. For
example, an increase in the number of stereocilia per bundle
(and by implication number of MT channels) will augment
the sensitivity of high-frequency hair cells. More interestingly, the reduction in the number of stereociliary ranks and
the greater increment between adjacent ranks in mammalian compared with nonmammalian auditory hair cells is
likely to minimize viscous resistance between adjacent
ranks and enhance the mechanical frequency response of
the bundle. In IHCs, the second and third ranks are also
much shorter than the tallest rank so the degree of overlap
between adjacent ranks is reduced, which might also minimize viscous resistance (FIGURE 2C). The most extreme example is seen in IHCs at the base of the bat cochlea (FIGURE
2D; Ref. 258), where the stereocilia are very short and the
number of ranks is reduced to two, the minimum possible
for gating MT channels. Such modifications in bundle morphology may be part of an evolutionary trend accompanying the expansion of the upper frequency limit of mammalian hearing. Changes in bundle height and stereociliary
number will also have consequences for hair bundle amplification and force-feedback onto the tectorial membrane
(35). The morphological diversity will be assessed again in
considering the MT channel currents, but it may signify a
matching of the accessory apparatus to the coding demands
during the course of evolution.
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The hair bundles in auditory organs of most vertebrates are
coupled to the tectorial membrane, an acellular 25- to 50␮m-thick sheet the motion of which provides the immediate
drive to the hair cells in vivo. In mammals, the tectorial
membrane consists of radial bundles of collagen embedded
in a matrix of glycoproteins, ␣-tectorin, ␤-tectorin, and otogelin (148, 210, 211). The mechanical stiffness of the tectorial membrane increases up to 20-fold from apex to base
(212) in parallel with the stiffness of the hair bundles, which
increases the shorter they are (21, 241). The importance of
the tectorial membrane structure for OHC excitation is
highlighted by the large loss in auditory sensitivity resulting
from targeted deletion of ␣-tectorin (147). The tectorial
membrane in nonmammals has a similar physical appearance and in reptiles such as the turtle, and in birds, the bundles are encased in pockets on the underside of the membrane,
strands of which are tightly attached to the tallest row of
stereocilia and to the kinocilium (90, 247). The connection
is different in mammals where OHCs of adults lack a kinocilium and the stereocilia in the tallest row are inserted into
individual pits visible in scanning electron micrographs of
the underside of the membrane (153, 227); the points of
attachment are associated with the protein stereocilin
(260). The mechanical strength of this attachment point is
unknown, but the histology suggests a tight coupling between the OHC bundles and the tectorial membrane so the
two vibrate in synchrony, which might be essential for forward transduction and for feedback amplification especially at high frequencies. The viscoelastic coupling to the
overlying membrane differs for IHCs, where a ridge known
as Hensen’s stripe on the undersurface of the tectorial membrane is closely apposed but not firmly connected to the
IHC bundles. It is usually supposed that fluid flow in the gap
between the tectorial membrane and the upper surface of
the organ of Corti is the stimulus for the IHCs (89, 114,
181) the bundles of which are therefore velocity-coupled to
basilar membrane motion. However, such fluid flow requires the edge of the tectorial membrane to be fixed to the
spiral limbus and not to float freely above the reticular
lamina. Mutation of the limbal anchoring protein otoancorin causes hearing loss thought to result from a failure in
the excitation of the IHCs (156).
III. MECHANOTRANSDUCER CURRENTS
A. Overview: Methods of Stimulation
Mechanoelectrical transduction in hair cells was first studied by recording receptor potentials with sharp microelectrodes (49, 109), and also more indirectly by measuring
extracellular microphonic currents (41, 42) in response to
manipulation of the hair bundle. More direct and precise
information about the MT channels was subsequently derived by patch-clamping either dissociated hair cells (11, 45,
46, 183) or cells in the whole epithelium (18, 54, 78, 96,
101, 114, 127, 139, 206, 233, 261). Recordings in the in-
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HAIR CELL MECHANOTRANSDUCER CHANNELS
tact epithelium generally yielded larger MT currents than
those from solitary hair cells, probably due to reduced mechanical damage to the hair bundle during tissue preparation. The initial work was done on nonmammals such as
frogs, turtles, and chickens, but more recent experiments
were performed on cochlear and vestibular hair cells of
mice, rats, or gerbils. Most MT channel properties, such as
ion selectivity, unitary conductance, and blocking agents,
are similar across species, but activation kinetics are faster
in mammals as might be expected because of the higher
frequency limit of their hearing. All demonstrate an inward
current at negative membrane potentials indicative of a
nonselective cation conductance elicited by deflections of
the hair bundle towards its taller edge.
An important difference between the reports is the type of
stimulator employed for deflecting the hair bundle. The two
main methods use either a fluid jet aimed at the bundle or a
stiff glass probe pressed against the bundle and driven by a
linear piezoelectric device. The fluid jet, although less destructive on the bundle, has the drawback that the force
generated is low-pass filtered, and it cannot impose movements with rise times faster than a few milliseconds (57,
139, 245, 261); moreover, the flow is directed at the entire
bundle and may therefore cause movements at the bundle’s
base as well as its tip. In the other method, a fire-polished
glass probe, a few microns in diameter, is placed against the
tallest or intermediate ranks of stereocilia. The piezoelectrically driven glass probe can generate very fast submillisecond bundle deflections but suffers from the disadvantage
that it may not contact all the stereociliary ranks causing
them to be differentially deflected. The method is also less
satisfactory for negative stimuli that pull the bundle back
towards its shorter edge (where the probe may lose contact
with the bundle). For mammalian hair cells, the glass probe
must be fashioned to fit the V-shape of the bundle (127,
233), a task difficult to implement with very short but wide
hair bundles such as those of IHCs in the mouse cochlea.
The fluid jet is good for assaying maximum MT currents,
whereas the piezoelectrically driven probe is more suitable
for measuring the kinetics of the MT current. In each case,
the potential limitations should be considered, with neither
method strictly reproducing the in vivo stimulus. A third
method, best approximating the physiological mode of
stimulation, has been to use a piezoelectrically driven glass
probe attached to the kinociliary bulb which is enlarged in
the frog saccule (101); this method has not been widely
employed because the bulb is small or the kinocilium absent
in most auditory hair cells.
The two main methods of stimulation yield similar values
for the maximum current (Imax), but differ in the range of
displacements encoded as seen in the representative results
for mouse OHCs (FIGURE 4). With step deflections of the
bundle using an attached glass probe, the inward current
developed rapidly and then adapted to a reduced level (FIGURE 4, A–C). The response was graded with bundle deflection, and the relationship between the peak current (I) and
displacement (X) could be described with a single (twostate) Boltzmann equation
I ⫽ IMAX ⁄ 兵1 ⫹ exp[(XO ⫺ X) ⁄ XS]其
(1)
where XO is the displacement to half activate the current
and XS is the slope factor. Use of the fluid jet (FIGURE 4, D
AND E) gave a similar maximum current and an I-X rela-
FIGURE 4. MT currents in mouse apical OHCs obtained using two different methods of hair bundle stimulation. A: deflections with a glass probe attached to a piezoelectric actuator, MT currents for step displacements of bundle, ⌬X, shown at top. B: relationship between the peak MT current and bundle displacement, ⌬X;
the experimental points were fitted with a single-stage Boltzmann equation with 10 –90% working range (WR)
of 280 nm. C: onset of MT current to a 130-nm step showing that the current develops with the same time
course as the stimulus but then undergoes fast adaptation with a time constant of 0.15 ms. D: hair bundle
stimulation with a fluid jet, MT currents shown below and bundle motion above; the smooth curve is the 40-Hz
sinusoidal driving voltage, and the noisy gray trace is the photodiode signal used to calibrate the movements.
E: current-displacement relationship for first cycle of response in D, fitted with a single-stage Boltzmann
equation with WR ⫽ 25 nm. In both sets of recordings, the holding potential was ⫺84 mV, and the Ca2⫹
concentration in the extracellular solution was 1.5 mM.
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
tionship also described by a single Boltzmann equation, but
with a 10-fold smaller slope factor XS, implying a substantially narrower working range. The precise form of the I-X
relationship has been interpreted as reflecting the channel
gating scheme (41, 45, 101). A single Boltzmann fit is expected for a two-state channel in which mechanical energy
modulates the transition between a closed state and an open
state
␤
closed↔ open
(2)
␣
with forward and backward rate constants, ␤ and ␣, respectively, depending on the force applied to the channel. The
channel opens and the current develops with a time constant ␣/␤. The open probability of the channel (PO) can be
specified as
PO ⫽ 1 ⁄ 兵1 ⫹ exp[Z(XO ⫺ X) ⁄ kBT]其
(3)
where Z, the single-channel gating force, is a measure of the
sensitivity of transduction, kB is Boltzmann’s constant, and
T is absolute temperature. Z can be related to ⌳, the range
of hair bundle displacements (166) over which the channel
open probability changes from 10 to 90%
⌳ ⫽ 4.4 · XS ⫽ 4.4 · kBT ⁄ Z
(4)
Displacements of the hair bundle are assumed to exert force
on the MT channel by extending a “gating spring” in series
with the channel (106), just as force can be applied by
stretching a macroscopic spring. The gating force can then
be expressed in terms of the microscopic stiffness (kg) of the
gating spring and channel motion in response to bundle
deflection, usually referred to as the gating swing (b), which
is the distance moved by the channel’s gate during opening
Z ⫽ ␥kgb
(5)
Typical values for the parameters can be derived experimentally on the basis of the gating spring model: stiffness of
the gating spring (kg) ⫽ 0.5–1.5 mN/m; the gating swing
(b) ⫽ 1– 4 nm; single-channel gating force (Z) ⫽ 0.25– 0.7
pN (21, 34, 106, 166, 168, 251). Occasionally, values outside this range have been required to reproduce experimental data, particularly for the gating swing (e.g., 5–11 nm;
Refs. 168, 251); such values seem unrealistically large if
representing a change in the dimensions of an ion channel
or the movement of a protein subcomponent. Also, larger
values for the gating spring stiffness are needed for mammalian cochlear hair cells (21, 251). The introduction of the
geometrical factor ␥ transforms the force at the tip of
the bundle to that occurring at the channel, with Z being the
single-channel gating force experienced at the tip of the
bundle. Because of the geometrical factor, forces are larger
and displacements smaller at the channel than they are at
the tip of the bundle.
958
While the single Boltzmann equation provides an adequate
approximation for many I-X relationships, some require a
(3-state) double Boltzmann equation to fit more accurately
the results. This formulation produces an asymmetric I-X
relationship with a curvature less pronounced at the upper
end than at the lower end of the sigmoid. A double-Boltzmann fit was needed to describe the I-X relationship in
nonmammalian hair cells (42, 45, 101) and in mammalian
hair cells (139) using both methods of bundle stimulation,
and has been linked theoretically to a small delay in the
current onset. The double-Boltzmann fit suggests a more
complex gating scheme with multiple closed-state transitions with mechanically sensitive rate constants, all consistent with a delay incurred by transition through the closed
states. While not questioning the validity of the doubleBoltzmann fits, it should be noted that not all I-X relationships have required a double-Boltzmann equation and
some, especially for cochlear outer hair cells, are adequately
described by a single Boltzmann equation (18, 96, 116). In
addition, there is a concern that the need for a double Boltzmann fit (and reduced curvature at the upper end) may
sometimes be an artifact of the mechanical stimulus, especially with an attached glass probe. The shallower corner
and greater working range at the upper end of the curve
could be partly due to a nonsynchronized deflection and
splaying of the stereociliary ranks, or to slipping of the
probe up the back of the bundle, both effects causing a
proportion of the MT channels to be recruited only for
larger stimuli. This is particularly the case for IHCs which,
because of their wide and flat hair bundles, are difficult to
stimulate, their I-X relationships often requiring a doubleBoltzmann fit (18, 233), but not always (114).
Whatever the method of stimulation, maximum currents of
several hundred picoamps up to several nanoamps (typically at ⫺80 mV holding potential) have been reported for
MT currents in a variety of hair cell types. It seems reasonable to assume that the largest currents are most representative of the in vivo state, since any damage to the cells or
bundles while the tissue is being prepared will be likely to
reduce the maximum current. For OHCs, the largest currents recorded at the apex are ⬃1.0 nA, and this amplitude
increases by up to twofold in progressing towards the highfrequency base (18, 96, 116, 130). A tonotopic gradient of
similar magnitude and polarity has been observed in auditory hair cells of turtle (205) and chicken (246), and in all
cases is thought to result from an increase in both the number of stereocilia per bundle, and the single-channel conductance (see sect. IVB). In contrast, no gradient has been
found in MT currents of IHCs (18, 23, 114).
B. Working Range of Mechanotransduction
The working range of hair cell transduction as specified by
“⌳” is important because it provides a measure of mechanical sensitivity and can be applied empirically to both two-
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HAIR CELL MECHANOTRANSDUCER CHANNELS
state and three-state Boltzmann fits. It can also be used to
derive a value for the Z (Eq. 4) in the two-state model.
Unfortunately, the substantial spread of values depending
on the mode of stimulation undermines precise analysis.
Nevertheless, several conclusions can be drawn from existing results. The narrowest working range, ⌳, inferred using
stimulation with an attached stiff glass probe is 0.2– 0.4 ␮m
for turtle auditory hair cells (206), frog saccular hair cells
(101), chicken auditory hair cells (246), rat apical OHCs
(18, 265), and mouse apical OHCs (233; FIGURE 4A); all
species listed have maximum bundle heights of 4 – 6 ␮m,
with the mouse being at the lower end of this range. Significantly narrower activation curves have been observed for
turtle (⌳⫽ 50 nm, Ref. 204), chicken (⌳ ⫽ 50 nm, Ref. 22),
rat OHCs (⌳ ⫽ 75 nm, Ref. 116), and mouse OHCs (⌳ ⫽
80 nm, Refs. 78, 219, 220; ⌳ ⫽ 32 nm, FIGURE 4B) when
force stimuli were applied with a flexible glass fiber or water
jet. Taken at face value, this discrepancy suggests that measurements with an attached stiff glass probe overestimate
the working range. A useful comparison is with the values
obtained in the hemi-cochlea preparation under more physiological conditions (96). Here, stimuli were delivered to the
basilar membrane, with force being transmitted to the bundles via displacements of the tectorial membrane; these
measurements gave ⌳ ⫽ 50 nm for basal OHCs and 190 nm
for apical OHCs, the stimulus in each case being expressed
as a displacement of the basilar membrane. With the assumption of a unitary gain between displacements of the
basilar membrane and the tip of the hair bundle (50), the
apical-basal difference largely reflects the fivefold shorter
bundle heights at the base. Thus the values of ⌳ for hair cells
in the hemi-cochlea are more similar to those with fluid jet
deflection of exposed bundles in rodent OHCs.
It is conceivable that the stiff glass probe causes more damage to the hair bundles than does the fluid jet, including
destroying or weakening interciliary links. However, the
simplest explanation for the difference between the two
methods of stimulation is that the stiff probe does not contact all stereociliary ranks equally so that when it advances,
the ranks are differentially recruited, thus smearing the
stimulus to the MT channels and broadening the I-X relationship. Such differential motion might occur along the
axis of symmetry so that the tallest edge of the bundle is
displaced more than the shortest edge. But laser imaging of
the two edges of bundles in frog hair cells argues against this
with all ranks moving synchronously (135). The motion
could also be distributed across the bundle so that the central stereocilia are displaced further than those on the periphery, producing a bell-shaped profile of displacement
amplitudes across the bundle. The latter problem will be
most acute for the mammalian cochlear hair cells where the
glass probe is unlikely to fit perfectly into the back of the
bundle, particularly if the shape of the bundle varies somewhat from one cell to another. The IHC bundles (with the
tallest stereociliary row being much longer than the two
shorter rows, and arrayed almost linearly) are likely to be
the most problematic so that deflections of the central stereocilia will pull the peripheral ones via lateral interciliary
links. Consistent with this notion, the I-X relationships of
IHCs are most asymmetric when assayed with a stiff probe
and exhibit a larger working range (⌳ ⱖ 400 nm; Refs. 18,
233, 238) than more physiological stimuli (⌳ ⫽ 60 –130
nm; Ref. 114). IHC bundles in vivo are probably stimulated
by fluid motion in the subtectorial space (89, 181), so use of
a fluid jet would provide the most physiological stimuli.
The value of ⌳ represents the displacements at the tip of the
bundle that modulate the MT channels through their working range. The ⌳ values assayed from fluid jet stimuli
(30 – 80 nm) may be taken as the best estimate for those
occurring in vivo, and can be compared with the movements of the basilar membrane that activate the MT channels through their working range as measured in intact animals. The latter can be inferred from the range of basilar
membrane movements over which the nonlinearity between
sound pressure and displacement is observed; this nonlinearity arises from OHC somatic contractions caused by the
receptor potentials generated by forward transduction.
With this idea, vibrations of the basilar membrane are only
amplified by OHC motility for displacements over which
the MT channel open probability varies. A number of complete measurements are available, and the amplitudes, interpolated from the upper extent of the nonlinearity, vary
inversely with the characteristic frequency (CF) of the location assayed. (The CF is defined as the sound frequency to
which the hair cells and auditory nerve fibers are most sensitive at a given position in the cochlea; variation in the CF
with location specifies the tonotopic map.) They extend
from ⬃100 nm at the apex (chinchilla CF ⫽ 600 – 800 Hz;
Ref. 200), through 30 nm for the mid-frequency region
(chinchilla: 6 –10 kHz; refs.199, 217) to 10 nm for the highfrequency region (gerbil: 35 kHz, Ref. 185; cat: 30 – 40
kHz, Ref. 38). Although no data are available for mouse or
rat, the range for other animals largely encompasses measurements on OHCs. Both imply that the operating range of
hair cell MT channels must be smaller in basal OHCs than
in apical ones, attributable to the shorter OHC bundles at
the base. About a fivefold difference in OHC bundle height
between base and apex has been reported for rat (216) and
chinchilla (153).
C. Kinetics of the MT Current: Activation
and Adaptation
Despite the limitations of the stiff probe for deflecting the hair
bundle, it has been the principal means of characterizing the
time course of MT current activation and adaptation. The
kinetics have been well documented for nonmammalian hair
cells, showing that in response to a step deflection of the bundle, the current develops with a submillisecond time constant
and then declines despite a sustained stimulus. The decline is
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
referred to as adaptation rather than inactivation because the
current can be recovered by increasing the stimulus amplitude;
the end result is a shift in the I-X relationship to larger displacements without change in shape or diminution in the maximum current (FIGURE 5; Refs. 61, 94, 233, 261). Adaptation
occurs over several distinct time frames from tens of microseconds to seconds. Two major processes are commonly recognized: fast adaptation operating on a millisecond or submillisecond time scale and slow adaptation acting over tens of
milliseconds (261, 270). A third process, conceivably metabolic and mediated by cAMP, may occur over a time scale of
seconds or more (206). The exact mechanisms underlying
these different forms of adaptation are still controversial, but
all may ultimately be regulated by intracellular Ca2⫹. Evidence
supporting this notion includes: 1) the fast adaptation time
constant (␶Af) is slowed by lowering extracellular [Ca2⫹],
(␶Af)⫺1 being proportional to Ca2⫹ influx via the MT channels
(207). 2) Adaptation is abolished by depolarizing to ⫹80 mV,
a membrane potential approaching the Ca2⫹ equilibrium,
which therefore minimizes Ca2⫹ influx (11, 46). 3) The position of the I-X relationship on the displacement axis is sensitive to the extracellular Ca2⫹ concentration and intracellular
Ca2⫹ buffering (94, 206, 209); as a consequence, the probability of MT channel opening at the bundle’s resting position
(PO,r) depends on external Ca2⫹ and internal Ca2⫹ buffering.
PO,r is determined from the ratio of the MT current active at
the bundle’s unstimulated position to the maximum MT current achievable with a saturating stimulus. It is an important
parameter that will be discussed in more detail later.
The prevailing hypothesis is that changes in stereociliary
Ca2⫹ concentration reset the range of bundle displacements
over which the MT channel is activated. Elevating the Ca2⫹
influx, as occurs during sustained channel opening, shifts
the I-X relationship positive to larger displacements causing
desensitization; on the other hand, reducing Ca2⫹ influx, as
may result from prolonged channel closure or lowering the
extracellular Ca2⫹ concentration, shifts the I-X relationship
in the opposite direction. The site(s) at which binding of
Ca2⫹ triggers adaptation are specific for this ion, and other
alkaline earth cations do not substitute for Ca2⫹ (94), even
though some, such as Sr2⫹, permeate the channel with comparable ease (183).
In terms of molecular mechanisms, it has been suggested
that slow adaptation involves a change in the attachment
point of the elastic element connected to the MT channels
(105). A specific proposal is that during a positive bundle
deflection, the upper end of the tip link slips down the side
of the stereocilium to reduce the tension in the elastic element (105). It is further suggested that the resting tension in
the tip link is maintained by an isoform of myosin (e.g.,
myosin Ic; Ref. 81) ascending the actin backbone of the
stereocilium. Support for this mechanism has come from
generation of transgenic mice expressing a mutant form of
myosin Ic that can be inhibited by intracellular ADP analogs
(100). Utricular hair cells from mice possessing the transgene Y61G-myosin Ic (in addition to wild-type myosin Ic)
demonstrated a slow adaptation (time constant ␶A ⫽ ⬃50
ms) that could be blocked by addition of the ADP analog to
the whole cell recording pipette; this maneuver also abolished any adaptive shift of the I-X relationship along the
displacement axis in the transgenic but not in wild-type hair
cells. These experiments were extended by constructing a
transgenic mouse in which only mutant Y61G-myosin Ic
was present, and showing that a faster form of adaptation
(␶A ⫽ 15–20 ms) in mouse vestibular hair cells was also
slowed or blocked by the ADP analog (234). While this
confirmed that a component of the adaptation employed
myosin Ic, it is unlikely that this component represents fast
adaptation; fast adaptation has a time constant of ⬍5 ms in
frog and mouse vestibular hair cells (261) and in auditory
hair cells of turtle (␶A ⫽ 0.5–3 ms; Refs. 45, 206) and rat
(␶A ⫽ 0.05– 0.5 ms; Refs. 127, 208). The millisecond time
constant of fast adaptation argues against its being mediated via an ATPase like that in any myosin isoform, which
FIGURE 5. Two-step analysis of fast adaptation in a rat
OHC. A: family of MT currents (controls) in response to
2-ms step deflections of the hair bundle with a glass probe
driven by a piezoactuator. B: MT currents for 2-ms step
deflections of the hair bundle superimposed on a 0.4-␮m
maintained adapting step; these stimuli were interleaved
with the control measurements as indicated by the time
axis at a stimulus repetition of 30 Hz. C: current-displacement (I-X) relationship for the control measurements (Œ)
and the adapted measurements (). Note the effect of
adaptation is to reset rapidly the working range by shifting
the I-X relation along the displacement axis by approximately the same amount as the adapting step; holding
potentials, ⫺84 mV. D: schematic of the effects of intracellular Ca2⫹ on the position of the I-X relationship along the
displacement axis. Elevated Ca2⫹, as occurs during adaptation, shifts the I-X relationship from its resting position,
denoted by dashed curve, to larger displacements. Reduction in intracellular Ca2⫹ during lowering external Ca2⫹ or
depolarization translates the I-X relationship to smaller displacements.
960
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HAIR CELL MECHANOTRANSDUCER CHANNELS
typically has cycling rates of ⬍20 s⫺1 (103) but is more
consistent with adaptation occurring at a site very close
(⬍30 nm) to the MT channel (45, 207, 270); a specific
hypothesis, based on fast adaptation being reflected in hair
bundle mechanics (203), is that it stems from a Ca2⫹-induced change in the force sensitivity of the MT channel
making the channel more difficult to open (34).
Despite the elegance of these experiments and the accompanying hypotheses, the precise mechanism of either kinetic
form of adaptation remains unclear. Furthermore, several
important questions linger particularly with regard to
mammalian cochlear hair cells. 1) There have been no studies on whether introduction of the ADP analog in Y61Gmyosin Ic mutants affects adaptation in cochlear OHCs; it
is conceivable that these cells are unusual and lack slow
adaptation, but it would be useful to provide definitive evidence one way or the other. 2) If the MT channels are
confined to the lower end of the tip link, as is now supposed
(19), myosin Ic motors localized at the upper end of the tip
link will have little access to Ca2⫹ entering via the MT
channel; how then are they regulated and what determines
the sliding of the attachment point with an increase in tension? 3) Immunolabeling has demonstrated that myosin Ic
is localized to both upper and lower ends of the tip links in
frog vestibular hair cells (76). However, no discrete foci for
myosin Ic are visible in cochlear hair cells, and labeling for
the myosin Ic isoform is distributed along the entire length
of the stereocilium (224). The diffuse labeling contrasts
sharply with that for three other myosins examined: myosin
IIIa and myosin XVa are both confined to the stereociliary
tips (224), whereas myosin VIIa clusters in a complex with
sans and harmonin-b at the upper end of the tip link (85).
Functional evidence for the role of myosin VIIa in cochlear
hair cells comes from the observation that several mutations
of this motor protein affect adaptation of the MT current
(138). Myosin VIIa climbing up the actin filaments in the
stereocilium may also be responsible for maintaining the
resting tip-link tension in mammalian OHCs (85). 4) Fast
adaptation may involve myosin XVa too (238). In IHCs of
shaker-2 mice lacking this isoform (Myo15 sh2/sh2), the stereocilia are short, and there is no increment in height between adjacent ranks of stereocilia. Large MT currents were
still recordable from IHCs of shaker-2 mice, but the current
lacked all signs of fast adaptation and was insensitive to external Ca2⫹. In contrast, fast adaptation and its dependence on
external Ca2⫹ remained intact in OHCs of shaker-2 mice.
The reasons for these differences are unclear. 5) Even the
role of intracellular Ca2⫹ in adaptation has lately been
questioned for cochlear hair cells (190), because none of the
three lines of evidence cited above for substantiating involvement of Ca2⫹ in adaptation is definitive in mammalian
cochlear hair cells. Step deflections of the hair bundle evoke
an MT current exhibiting fast adaptation (␶A ⫽ 45– 85 ␮s
depending on cochlear location), and these time constants
do slow on reducing extracellular Ca2⫹ from 1.5 mM to
(endolymphatic) 0.02 mM, but the increase in time constant
is small, no more than a factor of two (208). Depolarization
to ⫹80 mV approaching the Ca2⫹ equilibrium potential,
where Ca2⫹ influx ought to be minimal, diminishes the
extent of adaptation but never totally abolishes it, suggesting that either the remnant is Ca2⫹ insensitive or a stimulation artifact (e.g., slipping of the probe). Furthermore, in
recordings with pipette solutions containing high millimolar intracellular Ca2⫹, fast adaptation still occurred. Finally, lowering external Ca2⫹ to an endolymphatic concentration shifts the I-X curve leftward and increases PO,r to
⬃0.45, but whether this shift depends on the intracellular
Ca2⫹ buffer is contentious (20, 116, 190). Taken together,
these observations on adaptation in mammalian cochlear
hair cells do not fit with the simple notion, derived from
experiments on nonmammals, that adaptation, and the position of the I-X relationship are regulated by changes in
intracellular Ca2⫹.
A further puzzle is the potential involvement of cAMP in
mechanotransduction and adaptation (206), for which neither is the effect confirmed in mammals, nor is the signaling
pathway identified. An obvious route analogous to other
forms of adaptation might entail regulation by Ca2⫹: entry
of the ion through the MT channels could lead to activation
of a Ca2⫹/calmodulin-dependent adenylate cyclase (AC),
production of cAMP, and phosphorylation of the channel
or other protein linked to transduction. Of the nine membrane-bound isoforms, AC1 and AC8 are stimulated by
Ca2⫹ and AC5 and AC6 are inhibited by Ca2⫹, and there is
evidence for the occurrence of at least one of each category,
AC1 and AC6, in cochlear hair cells (59, 140, 172). Renewed interest in this signaling pathway has stemmed from
the recent discovery that a mutation in adenylate cyclase 1
(ADCY1) is linked to hearing imparimen in humans (223).
ADCY1 was found to be localized to the cochlea and vestibule of mice, including hair cells and their stereocilia
(223). Furthermore, mutation of the equivalent protein in
zebrafish caused a defect in hair cell mechanotransduction
(223). These results indicate the mechanism of MT channel
regulation by cAMP warrants further investigation.
The time course of MT current activation, as with adaptation, has been most thoroughly documented in nonmammalian hair cells (42, 45, 208). It is best quantified by recording in low external Ca2⫹ where distortion of the response onset by the development of adaptation is
minimized (208). Under these conditions, it was shown that
the activation time constant decreased with stimulus amplitude from 1 to ⬍0.1 ms, as might be expected if the mechanical stimulus energy modulates the MT channel opening and closing rate constants. In turtle auditory hair cells,
the activation time constant at low levels is roughly sixfold
faster than the adaptation time constant, so the two processes impose single-pole low-pass and high-pass filters, together creating a band-pass filter, albeit weak, on transduc-
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
tion. Because both time constants vary systematically with
distance along the basilar membrane, the center frequency
of the transduction filter approximately matches the CF of
the location (208).
It has been difficult to quantify the activation kinetics of the
MT current in mammalian auditory hair cells mainly because the kinetics are much faster than in nonmammalian
hair cells, and as a consequence, the speed of the piezoelectric stimulator becomes rate limiting. The turtle has a behavioral upper-frequency limit of ⬃0.6 kHz, so for MTchannel kinetics not to be limiting, the activation time constant should be no more than 0.25 ms, which is in range of
measurements. For the mouse, with an upper frequency
limit of 90 kHz, the corresponding time constant is ⬍2 ␮s.
Because of intrinsic under-damped oscillations in the conventional piezoelectric stimulator, the driving voltage must
be low-pass filtered at 10 kHz, achieving a rise time at
best of 30 ␮s (208, 233, 238). As a consequence, the MT
current onset kinetics are not resolvable, the rise time of the
current being contemporaneous with that of the stimulus.
However, a new solid state nanoscale cantilever has recently been developed that can produce deflections with
⬍10 ␮s rise time (58). Provided the recording bandwidth
can also be expanded, this stimulation technique promises
to improve measurements to the point where the kinetics of
cochlear hair-cell MT channels may be directly quantifiable.
D. Development of the Hair Bundle and MT
Currents
Precocial mammals such as guinea pigs are born with a
mature cochlea and able to hear at birth, but in altricial
rodents, like mice, rats, and gerbils, the auditory system, in
parallel with the visual system, does not develop fully until
2–3 wk after birth. Mice of the first two neonatal weeks in
age are often employed for hair cell study because their
temporal bones are not fully ossified, which enables cochlear access while minimizing mechanical damage. Hearing commences around postnatal day (P) 12, but a number
of properties are not fully mature until the end of the third
week (196). The hair bundle structure and mechanotransduction develop during the first week, and the final differentiation of IHCs and OHCs, with acquisition of adult
voltage-dependent ion channels (163, 164), OHC prestinbased electromotility (rat, Refs. 15, 184; mouse, Ref. 1),
maturation of the exocytotic machinery at the IHC synapse
(117), and pattern of differential innervation, (type I afferents to IHCs and efferents to OHCs, Ref. 196) proceeds
during the second and third weeks. The endolymphatic potential does not achieve its adult value until P16 (25, 235).
A critical juncture is the appearance of the tunnel of Corti,
the heads of the pillar cells separating the top surfaces of the
IHCs and OHCs, at around P11 just prior to the onset of
hearing. Even after the onset of hearing, auditory sensitivity
962
grows and does not reach an adult level in mice for several
months (63). These developmental changes have been reviewed in detail elsewhere (84, 196, 210), so only those
features linked to transduction will be described as they
bear on the properties measured in neonates that can be
generalized to the adult.
The distinctive arrangement of the OHC hair bundle develops during the first few postnatal days with emergence of
the three stereociliary rows out of a carpet of microvilli,
their elongation and formation of the staircase in heights,
and attainment of the characteristic “V” or arrowhead
shape (265). These morphological changes accompany acquisition of the MT current which achieves maximum amplitude by P2 to P4 in mice, hair cells at the base of the
cochlea preceding the apex by ⬃2 days (130, 149). Rats
exhibit a similar progression but are delayed about 1 day
relative to mice (265). The peak amplitude then remains
constant up until the onset of hearing (127), there being no
evidence of a further increase in the current into adulthood;
a rigorous test of this notion is not available as the experiments become increasingly difficult at later stages and the
current size tends to be underestimated. Despite no further
increase in the MT current amplitude, there are probably
subsequent variations in the molecular structure of the
channel. For example, the relative Ca2⫹ permeability of the
OHC MT channel decreases after P6 (130). The mechanism
of this transformation may be related to a change in the
expression of transmembrane channel like proteins, with
downregualtion of TMC2 after postnatal day 6 (see sect.
VIB and Refs 123, 130), but it occurs with no major change
in the magnitude of the MT current.
In altricial rodents there are also changes in the interstereociliary connections over the first two neonatal weeks (84,
91, 210). From birth, OHC bundles possess tip links (enabling mechanotransduction), but in addition they have ankle links and “transient lateral links,” including those coupling the tallest stereocilia to the kinocilium. A protein component of the ankle links is the very large G protein-coupled
receptor (VLGR1), which occurs along with the membrane
proteins usherin and vezatin (159, 172). VLGR1 and usherin are both defective in Usher syndrome type II (see sect.
VIA). In the cochlea, the ankle links and most lateral links
are eventually lost along with reabsorption of the kinocilium (196); the top connectors appear after P9, signifying
the mature condition (84, 260). Despite the transient appearance of the ankle links, mutations in VLGR1 are associated with early disorganization of the hair bundle and loss
of mechanotransduction in cochlear hair cells (159, 172). In
the vestibular organs, unlike the cochlea, the kinocilium
and ankle links are retained into adulthood. In the adult,
some lateral links between stereocilia in the same row must
persist to ensure bundle cohesion in both OHCs and IHCs
(253, 254). During early maturation of the bundle, as the
MT current achieves its full amplitude, a bidirectional re-
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HAIR CELL MECHANOTRANSDUCER CHANNELS
sponse is sometimes apparent, with MT currents being elicited by deflections both towards and away from the tall
edge of the bundle (172, 265). The origin of such bidirectional selectivity is unclear, but it usually disappears with
pruning of the lateral links during the first few days (265),
and may therefore be partly attributable to nonuniform
polarization of the tip links. Zebrafish lateral-line hair cells
also display a response to reversed-polarity bundle motion
early in development prior to formation of tip links. In this
case, the response has been ascribed to the presence of the
kinocilium since it disappears in specific mutants lacking
the kinocilium (133).
In charting early postnatal hair cell development in altricial
rodents, an important question is the extent to which a
functional MT channel is necessary for maturation of hair
bundle structure. Indeed, why does mechanotransduction
precede by more than a week the maturation of the bundle,
and the onset of hearing which is ultimately limited by
sound transmission through the middle ear? A clue may
derive from the observation that in mice with mutations in
proteins comprising the tip link and its attachments, the
bundles are deformed and, from P1 to P5, growth of the
stereocilia, especially those in the middle and shortest rows,
is stunted (28, 146, 271). Two possible mechanisms may
underlie this process (28, 250). First, the polymerization of
actin that is required for stereociliary elongation may be
stimulated by mechanical tension applied by intact tip
links to cytoskeletal proteins at the tops of the two
shorter rows of stereocilia. Alternatively, the rate of actin
polymerization might be increased by Ca2⫹ influx via
functional MT channels (250). Stereociliary Ca2⫹ handling matures during the early neonatal period in rodents
as indicated by upregulation of the plasma membrane
Ca2⫹-ATPase pump, which appears contemporaneously
with the onset of MT channel function (32). The Ca2⫹
pump, the PMCA2a isoform (60, 240), has fast kinetics,
is present at high concentrations in the stereociliary
membrane (32), and its absence in various mutants
causes hair cell degeneration and deafness (e.g., Ref.
240). Both mechanical stress and intracellular Ca2⫹ may
act cooperatively to regulate actin polymerization, so investigating the link between the two developmental
mechanisms in hair bundle maturation and stereociliary
growth will be an important line of investigation.
IV. REVERSE TRANSDUCTION
A. MT Currents In Vivo: MT Channel Set
Point and Hair Cell Resting Potential
Besides converting a sound stimulus into an electrical replica, auditory hair cells also participate in decomposing the
stimulus into its constituent frequencies recruiting several
mechanisms. In nonmammalian vertebrates, electrical tun-
ing of the receptor potential by voltage- and Ca2⫹-dependent K⫹ conductances is the foremost mechanism (8, 48,
66, 70, 151). However, because there is an upper limit to
the speed with which the K⫹ channels can be gated, this
mode of frequency analysis is restricted to frequencies below ⬃1 kHz (269), and in mammals, with their much
higher frequency range, another mechanism has evolved. In
the mammalian cochlea, passive mechanical tuning of the
basilar membrane is greatly amplified by the electromotility
of the OHCs (9, 10, 27) mediated by the membrane protein
prestin (51, 52, 276). The mechanical amplification attributable to OHC electromotility (the cochlear amplifier; Ref.
51) has often been referred to as an “active process” to
contrast it with the tuning bequeathed by the passive mechanics of the basilar membrane, and also because it is
susceptible to the metabolic state of the animal. In nonmammals, and also conceivably in mammals, a third mechanism, entailing tuned force generation by the stereociliary
bundle, may also operate (108). The performance of the
MT channels in vivo is critical for the operation of all three
tuning mechanisms.
Two features of the mammalian cochlea will augment the
MT current amplitudes in vivo relative to those measured in
isolated preparations. First, an endolymphatic potential
(EP) of ⬃100 mV in rats (25) and mice (235) and up to 80
mV in guinea pigs (80) will increase the electrical driving
force across ion channels in the stereociliary membrane facing the endolymph, thereby increasing the current for a
given MT conductance. Second, the MT channels are
blocked by millimolar external Ca2⫹ (42, 46, 207), a block
that is relieved by the low Ca2⫹ concentration in the endolymph bathing the bundles. Like the EP, endolymphatic
Ca2⫹ concentration varies with location (80). Normally,
isolated preparations are studied when they are totally immersed in an artificial perilymph resembling interstitial fluid
high in Na⫹ and Ca2⫹. The MT current increases by ⬃60%
when the external Ca2⫹ concentration is reduced from 1.5
mM to 20 ␮M, similar to that in mammalian endolymph
(26, 110). The increase is confined to negative membrane
potentials, with little change at positive potentials rendering
the MT current-voltage relationship more strongly inwardly rectifying (FIGURE 6B). The half-blocking concentration (KI) for Ca2⫹, whether applied extracellularly or
intracellularly (FIGURE 6, C AND D) is 1–2 mM (129, 207).
The similarity of KI for block by internal and external Ca2⫹
suggests a common binding site within the pore, and the
result differs from block by other larger antagonists such as
DHS and FM1– 43, which require much higher concentrations from the cytoplasmic aspect than the extracellular side to
block the MT channel. It should also be emphasized that
there are (at least) two Ca2⫹-binding sites associated with
the MT channel: one inside the channel that can block it and
a second elsewhere that mediates adaptation. These are different binding sites that probably have distinct selectivities
for the divalent ion.
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FIGURE 6. Rectification and Ca2⫹ block in MT
channels of mouse OHCs. A: apical OHC in which
hair bundles were deflected with a sinusoidal fluid
jet stimulus (top) superimposed on a voltage ramp
from ⫺150 to ⫹100 mV (middle), MT currents
being recorded in an extracellular saline containing 1.5 mM Ca2⫹ (perilymph) or 0.02 mM in
Ca2⫹ (endolymph). The continuous line through
the MT currents is the current-voltage response
in the absence of bundle stimulation; note that in
the low Ca2⫹, the resting open probability of the
channels (PO,r) is ⬃0.5. B: current-voltage relationships for an apical OHC in 1.5 mM Ca2⫹ and
0.02 mM Ca2⫹. The effect of reducing external
Ca2⫹ concentration is to remove block at negative
membrane potentials and produce a more
marked inwardly rectifying relationship. C: block
of MT channel as a function of the extracellular
Ca2⫹ concentration. Mean ⫾ SE of the MT current at ⫺84 mV is plotted against the Ca2⫹ activity; intracellular solution contains 142 mM CsCl
and 1 mM EGTA. Points fitted with a Hill equation
with half-blocking concentration, KI ⫽ 0.9 mM
and Hill coefficient, nH ⫽ 1.0. D: block of MT
channel as a function of intracellular Ca2⫹ activity.
Mean ⫾ SE of the MT current at ⫺84 mV is
plotted against the Ca2⫹ activity; extracellular solution contains 160 mM NaCl and 0.02 mM
Ca2⫹. The lowest intracellular Ca2⫹ activity is
equivalent to 1 mM EGTA. Points fitted with a Hill
equation with KI ⫽ 2.5 mM and nH ⫽ 1.0.
Owing to the Ca2⫹ regulation of adaptation, a second
consequence of exposure to low Ca2⫹ endolymph is an
increase in the resting open probability of the MT channels. This is attributable to a shift of the I-X relationship
in the negative direction, hence increasing the probability
of the channel being open at the resting (nonstimulated)
bundle position (PO,r). A high resting open probability
is seen across vertebrate classes when the hair bundles
are exposed to low Ca2⫹ concentration similar to endolymph; for example, PO,r ⫽ 0.28, turtle (65, 209);
0.28, chicken (246); and 0.46, rodents (20, 116). The
increase in PO,r generally depends on the intracellular
calcium buffer concentration, being larger with stronger
buffering, which rapidly removes the Ca2⫹ from the internal face of the channel (209, 214). To estimate PO,r
under conditions resembling those in vivo, it is necessary
to make hair cell recordings with perforated-patch electrodes for which low-resistance electrical contact is made
to the cell interior without eluting the Ca2⫹-binding proteins. The main mobile calcium buffers include calbindinD-28k, calretinin, and parvalbumin-␤ (also named oncomodulin in mammals), which are present in the soma
and the hair bundle at up to millimolar concentrations
(62, 92, 93, 182, 246, 273).
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A high PO,r in vivo will maximize sensitivity and minimize
distortion. For pure-tone sinusoidal stimuli, the MT current
will be generated close to the point of maximum slope of the
I-X relationship and will be approximately symmetrical
about rest, increasing on one half-cycle and decreasing by
an equal amount on the other half-cycle of the sinusoid. For
low level stimuli, transduction is approximately linear,
thereby minimizing distortion. But another more significant
consequence is that, in the absence of stimulation, a standing inward current will flow through the partially open MT
channels to depolarize the hair cell. This may be termed a
“silent current” (116, 279) by analogy with the dark current in photoreceptors, and results in a resting potential of
⫺40 to ⫺45 mV well depolarized from the K⫹ equilibrium
potential. In the turtle auditory papilla, this is essential to
optimize electrical tuning by setting the membrane potential near that required for activating the voltage-sensitive
Ca2⫹ and Ca2⫹-activated K⫹ channels (65). As a consequence, the quality factor of electrical tuning is maximal at
rest. In mammalian OHCs, the standing current adjusts the
membrane potential to be near the half-activation of the
somatic electromotility underscored by prestin, thereby optimizing the somatic motor feedback (116). In both reptile
and mammal, both the inward MT current in the stereocilia
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FIGURE 7. Tonotopic variation in the MT conductance and K⫹ conductance in rodent OHCs. A: schematic of an
OHC illustrating the two principal ionic currents that determine the resting potential. An inward current (IMT) flows
through open MT channels, the electrical driving force resulting from the positive endolymphatic potential (EP ⫽
⫹90 mV) and the negative resting potential, ⫺40 to ⫺50 mV. The MT channels are partially open due to a low
endolymph Ca2⫹ of 0.02 mM. B: maximum MT conductance (GMT) in endolymph Ca2⫹ as a function of the CF of the
cochlear location: , gerbil; , rat. C: maximum voltage-dependent K⫹ conductance (GK) as a function of the CF of
the cochlear location: , gerbil; , rat. The K⫹ current in hearing animals older than P12 is dominated by IK,n flowing
through KCNQ4-containing channels in the OHC basolateral membrane. Measurements are from Johnson et al.
(116), extrapolated to the in vivo condition including a body temperature of 37°C.
and the outward voltage-dependent K⫹ current in the basolateral membrane increase in amplitude along the tonotopic axis towards its high-frequency end (Refs. 8, 116;
FIGURE 7), the current amplitudes being approximately
matched to maintain a constant resting potential. In birds
too, the MT current increases tonotopically reflecting increases in single-channel conductance and number of stereocilia per bundle (Ref. 146; FIGURE 8).
FIGURE 8. Tonotopic variation in the MT currents in hair cells of the chicken auditory papilla. A: MT currents in
three short hair cells in response to fluid jet deflections of the hair bundle; holding potential is ⫺84 mV. Sinusoidal
driving voltage to the fluid-jet is shown at the top. Cell locations in the epithelium given beside traces as the fractional
distance (d) from the apical end of the papilla (d ⫽ distance from the apex scaled by the total length of papilla, ⬃3.6
mm). B: collected peak amplitudes of MT currents versus location for tall and short hair cells at 33°C. Line is
exponential fit to all points. Tonotopic variation in the number of stereocilia per bundle (crosses referred to the right
hand axis) replotted from the data of Tilney and Sauders (248). C: MT current per stereocilium (continuous line)
derived from the data in B by dividing the fit to the experimental MT currents by the fit to the number of stereocilia
per bundle. MT current per tip link (dashed line) derived by dividing the continuous line by 0.88, the approximate
ratio of tip-links to stereocilia in a chicken hair bundle. [Modified from Tan et al. (246).]
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
A further outcome of the tonotopic gradient in conductances in OHCs is a systematic decrease in the membrane
time constant (␶m) along the cochlea so that the frequency of
the “membrane filter” (1/2␲ ␶m) approximately matches the
CF of the location, thus minimizing low-pass filtering of the
receptor potential (116, 179). Thus it is expected that
the receptor potential will faithfully reflect the MT current up
to high frequencies, ensuring voltage-dependent gating of
prestin on every cycle of the stimulus. Such cycle-by-cycle
activation of prestin is a necessary requirement of this process to amplify the vibrations of the cochlear partition. Thus
a 200-Hz acoustic stimulus is expected to elicit a 200-Hz
modulation in membrane potential around the resting potential, as should a 20-kHz stimulus. In the mammalian
cochlea, such behavior is confined to OHCs, whereas in
IHCs there is no evidence for a tonotopic gradient in either
the MT current or the membrane time constant, and the cell
resting potential is more hyperpolarized at ⫺55 mV (116,
137). The IHC membrane time constant is ⬃0.25 ms, and
the corner frequency of the equivalent low-pass filter is 600
Hz (116, 137, 186). Above this frequency, the sinusoidal
component of an asymmetric receptor potential will be attenuated to produce a sustained depolarization known as
the summating potential (186, 219). This transformation
ensures that the signal (a steady depolarization) is transmitted by the synaptic machinery, which otherwise would filter
out a high-frequency sinusoidal signal due to the time
course of exocytosis and neurotransmitter release.
B. Hair Bundle Mechanics and Amplification
The MT channels are also implicated in amplification
through their involvement in force generation by the stereociliary bundle. This property reflects reversibility in MT
channel activation, an applied force opening the channels
and channel gating generating a force output. The phenomena have emerged from study of hair bundle mechanics in
both mammalian and nonmammalian hair cells. To characterize hair bundle mechanics, as with other mechanical systems, it is necessary to apply a force to the system and
observe the resulting displacement. Calibrated force stimuli
can be delivered to the tip of the hair bundle using a fine
glass fiber, fabricated to be more flexible (i.e., have a smaller
stiffness) than the bundle (49, 104, 105, 241). (The flexible
fiber is equivalent to a spring, and its spring constant is
determined by observing the bending produced by weights
hung from the tip.) The deflection of the hair bundle is
derived by projecting an image of the bundle onto a pair of
photodiodes and monitoring the change in photocurrent
as the shadow of the bundle, or the attached fiber, traverses
the photodiodes (49). Such measurements have shown that
the hair bundle does not behave like a simple spring: its
mechanical stiffness is compound, with both a passive linear component and an active, nonlinear component (106,
204, 220, 256). The observation of a nonlinear component
when first reported was surprising as it depended on the
gating of the MT channels (106).
966
The passive stiffness arises from the parallel combination of
the stiffness of the stereociliary pivots, the interciliary links
(especially the tip links), and the MT channel gating springs
(192). The pivot accrues from bending of the the densely
packed bundle of actin filaments, cross-linked by TRIOBP,
at the stereociliary rootlets (134). The presence of the crosslinking protein TRIOBP is essential for proper hair-bundle
function, and when this protein is mutated, the stereocilia
lack rootlets and become much easier to deflect and damage, leading to profound deafness (134). The passive stiffness also includes a contribution from the gating springs
connected to active MT channels, and it therefore has a
larger value in hair cells having more functional channels.
Measurements on hair cells with large MT currents have
yielded passive stiffness values of 1–5 mN/m in frog saccule
(106), turtle auditory papilla (49, 204) and apical OHCs of
mouse (78) and rat (21), all of which have bundles of similar
height (4 – 6 ␮m). The passive stiffness increases in proportion to the number of stereocilia and inversely with the
square of the bundle height (49, 104), so hair bundles of
basal OHCs, composed of more and shorter stereocilia, will
be substantially stiffer than those of apical OHCs (241).
Destruction of the tip links, and hence loss of the gating
springs, by treatment with low-calcium BAPTA, reduced
the stiffness of frog hair bundle by ⬃30% (113) and rat
OHC bundles by 40% (21). That portion of the bundle
stiffness contributed by the gating springs represents the
fraction of work done in deflecting the bundle that is funnelled through the gating springs to open the channels. This
30 – 40% fraction implies that the stimulus energy is efficiently coupled to the MT channel.
The effect of channel gating is to generate a force in the
same direction as the imposed displacement, the gate flipping open under tension from the tip link, which effectively
reduces bundle stiffness. This component of bundle stiffness
is therefore nonlinear and can be shown to exhibit a Gaussian dependence on PO, the open probability of the MT
channels. The stiffness of the bundle decreases as the channels open, reaching a minimum at the point where PO is
⬃0.5, and then increases again at larger open probabilities
(106). The nonlinearity has been extensively documented in
frog saccular hair cells, and it is abolished, producing an
increase in bundle stiffness, by blocking the MT channels
with dihydrostreptomycin (106). Although the nonlinearity
is less prominent in mammalian cochlear hair cells, the stiffness of rat OHC bundles also increased significantly by
blocking the MT channels with dihydrostreptomycin (21).
The relative magnitudes of the passive and active components determine the extent of the nonlinearity. For some
hair bundles, the stiffness is dominated by the active component to the extent that, over the central range of channel
gating, the stiffness can become negative, a feature that has
been linked to spontaneous oscillations of the bundle (167,
251). The more usual behavior is that the bundle stiffness
remains positive over the entire operating range of trans-
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HAIR CELL MECHANOTRANSDUCER CHANNELS
FIGURE 9. Active force generation in turtle auditory hair
cells. A: one model of the MT channel fast adaptation. The
channel is anchored to the tip link and to the cytoskeleton by
springs, GS and GS’, respectively. Bundle deflection causes
force to be applied via the tip link. This extends the spring
and opens the channel allowing influx of Ca2⫹ which binds to
the channel and/or the cytoskeletal spring causing channel
reclosure. Closing of the channel exerts force on the tip link
which pulls the bundle back in the negative direction. B: a
force step applied to a turtle auditory hair cell bundle with a
flexible fiber elicits a positive bundle deflection (monitored by
imaging the bundle on a photodiode pair) and an inward MT
current. As the current adapts, the bundle moves back in
the negative direction, with a parallel time course. C: the
time constant of the hair bundle recoil decreases with hair
cell location along the papilla towards the high frequency
end. [Modified from Ricci et al. (203).]
duction (106, 204, 220). Nevertheless, whenever there is a
substantial nonlinear component, factors affecting the
probability of opening of the MT channels, such as adaptation, will effectively alter the stiffness of the bundle, and
may cause it to move (17, 203; FIGURE 9). This is often
referred to as “active bundle motion,” by analogy with the
active amplification process in the mammalian cochlea, because it is powered by energy supplied from the hair cell.
Since adaptation is thought to be driven by influx of Ca2⫹
and its interaction with the MT channels, a source of energy
for the active bundle motion may derive from the large
gradient in Ca2⫹ concentration across the stereociliary
membrane. Since the time constant of fast adaptation in
auditory organs decreases inversely with CF, the time constant of active hair bundle motion will also vary tonotopically (FIGURE 9), and it is therefore conceivable that active
mechanical feedback contributes to frequency selectivity.
Active hair bundle motion attributable to channel gating
has been amply documented in isolated hair-cell epithelia in
which the overlying gelatinous otolithic or tectorial membrane has been removed, but whether it is significant, or is
employed to amplify and tune hair cell responses in an
intact auditory end organ, is debatable. One immediate difference between the two types of hair-cell preparation is
that the spontaneous bundle oscillations observed in isolated epithelia as a manifestation of the active process (167)
are suppressed when a gelatinous membrane is left attached
to the hair bundles (242). The best case for a functional role
for active bundle movements has been made for the abneurally placed short hair cells of the avian cochlea, which by
their position and meager afferent innervation resemble
OHCs of the mammalian cochlea. It has been proposed that
their function, similar to OHCs, is to augment the vibrations of the tectorial membrane, thereby modifying the input to the tall hair cells, the avian equivalent of the IHCs,
which contact the majority of the afferent nerve fibers
(160). Although tall avian hair cells possess electrical tuning
(70, 246), this mechanism may not extend to the upper limit
of bird hearing, 5–10 kHz (66, 269). Modeling the contribution of active bundle motion to frequency tuning (employing a scheme that involves Ca2⫹ binding to the MT
channel) has suggested that, with the distribution of avian
hair bundle morphologies, resonant frequencies from 0.05
to 5 kHz might be achievable (35, 244); the predicted range
was partly dictated by altering the number of stereocilia,
and hence number of MT channels per bundle (248). In the
chicken, as in other vertebrates, the MT current increases
along the tonotopic axis because of increases in both singlechannel conductance and numbers of stereocilia (FIGURE 8),
and the two factors together might in theory increase the
speed of force generation at higher CFs (FIGURE 9). However, recent experimental measurements of active hair bundle movement in chick auditory hair cells have indicated
that besides any processes driven by MT channel gating,
there is an equal component attributable to voltage-dependent activation of prestin, as in the mammalian cochlea
(22). At the very least, the two processes, hair bundle motility and prestin-driven somatic motility, might act synergistically, with the latter becoming dominant towards the
higher frequencies.
It remains an open question as to whether force generated
by MT channel gating and adaptation supplements the con-
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
tribution of prestin to amplification and frequency analysis
in mammalian cochlear hair cells (51, 108). There are indications that OHC bundle mechanics possess a nonlinearity
linked to fast adaptation (21, 126, 220), and moreover
manipulations that interfere with adaptation can alter the
mechanics of the cochlear partition (29). In addition, OHC
depolarization evokes hair bundle movements due both to
prestin (115, 128) and to MT channel gating (128). Most of
these studies have employed isolated hair-cell epithelia with
unencumbered hair bundles. However, there is sparse evidence for a role of active hair bundle motility in vivo where
the viscoelastic behavior is complicated by the OHC bundles being tightly attached to and loaded by the tectorial
membrane, which is affixed to the sidewall of the cochlea
(156) and contributes significant additional stiffness as well
as mass. In contrast, both targeted deletion of prestin (152),
and mutations that diminish its physiological activation
(52), abolish OHC contractility and produce a 100- to
1,000-fold loss of auditory sensitivity in vivo. It is, however,
difficult to isolate experimentally the contribution of the
MT channels in vivo, since forward transduction is obligatory to generate the receptor potentials needed for activating prestin. This difficulty is illustrated by the recent attempt to eliminate active hair bundle motility in vivo by
injecting Ca2⫹ into the endolymph. The ensuing loss of
sensitivity and nonlinear compression in the basilar membrane vibrations were interpreted as due to Ca2⫹-dependent effects on MT channel adaptation (180). But these
effects could have equally well resulted from diminished
activation of prestin secondary to changes in OHC resting
and receptor potentials. Thus elevating endolymphatic
Ca2⫹ will both block the MT channels and reduce their
resting open probability, causing a decrease in the receptor
potential amplitude and a hyperpolarization of the OHC,
respectively. At this stage, more experiments are needed to
establish unequivocally whether active hair bundle motility
has a significant role in the mammalian cochlea.
V. THE MECHANOTRANSDUCER CHANNEL
PROPERTIES
A. MT Channel Pore Properties: Ion
Selectivity and Block
In the absence of a molecular composition, measurements
of ion conduction through the MT channel pore can provide some insight into the channel structure. The native MT
channel is a nonselective cation channel with high permeability to Ca2⫹. Various groups have made partial measurements (40, 46), but the most complete study was performed
on chicken vestibular hair cells (183) for which the ion
selectivity was inferred from reversal potential measurements. The permeability sequence for monovalent cations is
Li⫹ ⬎ Na⫹ ⬎ K⫹ ⬎ Rb⫹ ⬎ Cs⫹. The permeability varies
inversely with the ionic radius and is equivalent to the
968
Eisenman sequence XI (98), indicating the obstacle to permeation is not ion dehydration but strong interaction with
negative charges in the selectivity filter of the pore. The MT
channel in chicken vestibular hair cells is the only one for
which a comprehensive permeability sequence has been obtained, but a sequence in turtle auditory hair cells derived
from the relative amplitudes of MT currents in different
external solutions is Cs⫹ ⬎ K⫹ ⬎ Na⫹ ⬎ Li⫹, the reverse
order to the permeability sequence inferred from reversal
potentials (64). The opposite order for the two sequences is
inconsistent with a simple channel model occupied by a
single ion (98), and requires binding and interaction of multiple ions within the pore. A corroborate finding is the
anomalous mole fraction for frog hair cell channels exposed
to mixtures of Ca2⫹ and Na⫹, which displays a nonmonotonic change in current with systematic increase in concentration of one of the components of the mixture (157).
Similar properties, in terms of selectivity sequences and
anomalous mole fraction, were found for anion permeation
through glycine and GABA receptor channels and were
modeled with two ion binding sites (24). Because MT channels in different end organs may have distinct subunit compositions and properties, it would be desirable to measure
all three properties (permeability and conductance ratios
and anomalous mole fraction) on a single cell type such as
the mammalian OHC.
The MT channel of chicken vestibular hair cells also conducts divalent cations with a permeability sequence of
Ca2⫹ ⬎ Sr2⫹ ⬎ Ba2⫹ ⬎ Mn2⫹ ⬎ Mg2⫹ (183), all being more
permeable than K⫹ or Cs⫹, e.g., PCa/PCs ⫽ 4.7. Unlike
monovalent cations, the sequence does not follow ionic size,
and the impermeability of Mg is atypical. Since the influx of
Ca2⫹ is important for MT channel adaptation, its permeability has been well studied in several cell types under various conditions (18, 40, 130, 157, 207), and in all cases, a
high permeability for the divalent was found. In the mammalian cochlea, the permeability differs between OHCs and
IHCs with PCa/PCs ⬃4.5 and 6, respectively, implying differences in the pore region between these two cell types (18,
130). For the OHCs there are also changes in PCa/PCs with
development (130). In extracellular solutions containing
1.5 mM Ca2⫹, the divalent ion carries ⬃12–15% of the MT
current (18). The high permeability to Ca2⫹ must be associated with stronger binding of the divalent ion in the pore,
and as a consequence, Ca2⫹ lingers around in the pore and
can block the current carried by monovalent ions, the halfblocking concentration (KI) being 1–2 mM (129, 208). The
importance of this KI value is that in vivo, when the hair
bundles are immersed in endolymph containing 0.02 mM
Ca2⫹, channel block is fully relieved, thus maximizing the
monovalent current carried by K⫹ while allowing influx of
sufficient Ca2⫹ to mediate adaptation. Under such conditions, the channel’s current-voltage relationship displays
strong inward rectification, even with identical monovalent
ions on each side (188), indicative of an asymmetric pore
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HAIR CELL MECHANOTRANSDUCER CHANNELS
configuration. Such asymmetry is also evident in the permeation of large organic cations and blocking agents (64, 165,
257). In acting as a permeable blocker for the channel, Ca2⫹
resembles other larger positively charged antagonists such
as dihydrostreptomycin and FM1– 43.
The MT channel also exhibits substantial permeability to
organic cations, even large ones such as choline, tetramethylammonium (TMA). and tetraethylammonium (TEA) ions
which have permeabilities relative to Cs⫹ (PX/PCs) of 0.33,
0.20, and 0.17, respectively (183). Such high permeability
to organic ions is unusual among ion channels (98) and
suggests a large-diameter pore. More systematic measurements using a range of organic cations with different dimensions have estimated that the diameter of the narrowest part
of the pore when it is accessed from the extracellular face is
greater than 1.2 nm (64). In contrast, when organic cations
were introduced into the intracellular solution to be used
for carrying outward current at positive membrane potentials, the apparent diameter of the pore was only 0.6 nm
(188). This again suggests an asymmetric pore, having an
enlarged extracellular vestibule to funnel large cations into
the pore, and a smaller intracellular face for which elongated organic cations must adopt an “end on” configuration to gain access to the pore. A similar model has emerged
from quantifying and modeling the voltage dependence of
blocking agents (165, 257).
Current flow through the MT channel is inhibited by inorganic polyvalent cations such as Gd3⫹, La3⫹, and Ca2⫹, all
of which block other mechanosensitive channels, as well as
an assortment of organic polycations (64; TABLE 1). The
most efficacious nonpeptidergic blockers are FM1– 43 and
curare, with half-blocking concentrations (KI) of ⬃2 ␮M
when applied extracellularly. The highest affinity is
achieved with three polycationic peptides all of which block
at submicromolar concentrations. For example, HIV-TAT
is a 12-residue peptide, of which 8 are basic arginines or
lysines, and has a half-blocking concentration of ⬃27 nM
(56). Another class of basic peptide is GsMTx-4, which is a
34-residue peptide toxin from the tarantula spider Grammostola spatulata and has a half-blocking concentration of
⬃0.6 ␮M (23). The interesting feaure of this peptide is that
it is known also to block mechanosensitive (stretch-activated) channels in various tissues (243). A prominent feature of most blocking compounds is the voltage dependence
of their action and, although the exact voltage sensitivity
differs between the compounds, all are most effective at
hyperpolarized membrane potentials where they may be
expected to be swept into the pore from the outside. The
voltage dependence occurs with most of the smaller molecules and even extends to the peptides.
The voltage dependence of block has been quantified experimentally for the aminoglycoside antibiotic dihydrostepto-
Table 1. Some blocking agents of the hair cell’s mechanotransducer channel
Blocking Agent
Ca2⫹
Gd3⫹
La3⫹
Diltiazem
Amiloride
Amiloride
Amiloride
Benzamil
Dihydrostreptomycin
Dihydrostreptomycin
Dihydrostreptomycin
Dihydrostreptomycin
Dihydrostreptomycin
Quinine
Ruthenium red
Curare
Curare
FM1-43
GsMTx-4§
D-JNK1, D-HIV-TAT§
KI, ␮M
1,000
10
4
228
50
53
24
6
8 (0.25 mM Ca2⫹)
44 (5 mM Ca2⫹)
23 (2.5 mM Ca2⫹)
14, 75‡ (2.8 mM Ca2⫹)
7* (1.3 mM Ca2⫹)
10
3.6
2.3
6.3–16
2.4†
0.65
0.01–0.07, 0.03
nH
1
1.1
0.7
1.8
1.7
2.2
1.6
1
0.9
0.96
1.2
1.4
1.0
2.1
1.2*†
0.92
References
Ricci and Fettiplace (207); Kim et al. (129)
Kimitsuki et al. (131)
Farris et al. (64)
Farris et al. (64)
Jørgensen and Ohmori (119)
Rüsch et al. (218)
Ricci (202)
Rüsch et al. (218)
Kroese et al. (136) (frog)
Kroese et al. (136) (frog)
Kimitsuki and Ohmori (132) (chicken)
Ricci (202) (turtle)
Marcotti et al. (165) (mouse)
Farris et al. (64)
Farris et al. (64)
Glowatzki et al. (82)
Farris et al. (64)
Gale et al. (75); Meyers et al. (171)
Beurg et al. (23)
Desmonds et al. (56)
KI is the half-blocking concentration, and nH is the Hill coefficient. *Value at ⫺84 mV is given. For some
blockers, such as dihydrosteptomycin, KI depends on membrane potential and block is relieved at positive
potentials. †KI and nH given for ⫺104 mV. ‡KI values in low-frequency and high-frequency cells quoted. §Small
peptides, 12–34 residues.
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
mycin (DHS) (136, 165, 183), the fluorescent dye FM1– 43
(75), and the diuretic amiloride (218), and each has been
modeled using rate theory to deduce the energy profile
along the pore (257). For the divalent DHS, a good description of the block was achieved with a single negatively
charged binding site ⬃80% of the way across the energy
profile with energy barriers at the extracellular and intracellular extremities of the pore. The intracellular energy
barrier is larger, preventing access of the blocking molecules
from the cytoplasmic face, so the half-blocking concentration for DHS is at least 100-fold larger from the inside than
from the outside (165, 257). When presented at a high
intracellular concentration, DHS does block, but only at
positive membrane potentials, indicating that the binding
site can be accessed from the internal face of the pore, and
supporting the notion that DHS is a permeant blocker.
Channel block by DHS also depends on the Ca2⫹ concentration (TABLE 1), the affinity being higher in reduced external Ca2⫹ (TABLE 1) suggesting that DHS and Ca2⫹ compete
for the same binding site. FM1– 43, like DHS, is divalent
but blocks with a Hill coefficient of ⬃2 at intermediate
membrane potentials, indicating two drug molecules are
needed to block the channel (75), and suggesting that
FM1– 43 competes with Ca2⫹ for a minimum of two binding sites about halfway through the pore of the MT channel.
However, it should be noted that the Hill coefficient for
FM1– 43 binding is voltage dependent and at hyperpolarized potentials of ⫺104 mV, nH ⫽ 1.2 (75). Since FM1– 43
is an elongated molecule with a maximum end-on diameter
of ⬃0.8 nm and a length of ⬃2.2 nm, this sets a minimum
diameter of ⬃0.8 nm for the pore; moreover, the vestibule
facing the extracellular side of the channel must be sufficiently large to accommodate the heads of at least two
FM1– 43 molecules (257). Block of MT channels by the
diuretic amiloride and its derivatives, which have a Hill
coefficient of ⬃2 (218), could also be modeled with a negatively charged binding site within the pore at 0.55 and 0.45
of the way across the energy profile for amiloride and benzamil, respectively (257). It is not clear why the location of
the binding site is different for the amiloride diuretics and
for DHS, although the value refers to the distance across an
energy profile and not a physical distance.
Similar to Ca2⫹, both FM1– 43 and DHS are permeant
blockers that can traverse the pore and accumulate within
the hair cell, a property with significant implications. Since
FM1– 43 is fluorescent when bound to membranes, the
lighting up of hair cells on exposure to external FM1– 43
provides a good test of functional MT channels (75, 171); it
is therefore a useful assay for transduction defects in mutants without recourse to single-cell recordings (e.g., Ref.
123). This assay assumes that in a hair cell under normal
conditions, some of the MT channels are open at rest and
also that there are no other routes by which FM1– 43 enters
the cytoplasm, ignoring the fact that it can accumulate via
clathrin-coated pit endocytosis. In particular, in using
970
FM1– 43 as an assay for mechanotransduction, it is important to perform the appropriate controls to eliminate other
access routes to the cytoplasm, such as endocytosis, which
has been described for FM1– 43 in cochlear hair cells (86).
Intracellular accumulation of aminoglycoside antibiotics is
important because it culminates in hair cell death and apoptosis (213) possibly by targeting the mitochondria. There
has been considerable debate over whether DHS can enter
the hair cells via other pathways, but the current consensus
is that the predominant access route is via the MT channels
(4, 264).
Several lines of evidence, including assays of pore size with
organic cations, the asymmetry of inhibition by certain
blocking compounds, and the inwardly rectifying currentvoltage relationship, point to the MT channel being asymmetric between its external and cytoplasmic faces. The pore
has been hypothesized as possessing a wide external vestibule, narrowing down to a conduction path that embodies
the selectivity filter (FIGURE 10; Refs. 18, 188, 257). A specific model for the pore represents it energetically with one
or two negative-binding sites, and a large intracellular energy barrier. It is instructive to compare models of the MT
channel with the cyclic nucleotide-gated (CNG) channel,
which has similar physiological properties, but also a
known molecular structure (122), so it is possible to relate
binding sites in the two types of channel. The CNG channel,
underlying transduction in the photoreceptors and olfactory receptors, is a nonselective cation channel with a high
permeability to Ca2⫹ (PCa/PNa ⫽ 6.5 for rods). Ca2⫹ is also
a permeant blocker (KI ⫽ 0.3 mM in rods), whose antagonistic behavior is abolished by mutating a single glutamate
in the pore region (E363 in rods; Refs. 122, 215). Recent
structures of CNG-like channels (55) crystallized in K⫹ or
in Ca2⫹ indicate multiple negative binding sites in the pore.
Ca2⫹ binding in NaK2CNG-D is more complex, and the
difference map reveals four major peaks along the ion conduction pathway: three at sites inside the filter and one
within the funnel or vestibule just above the external entrance. If these features of the CNG channel resemble the
MT channel, the arrangement is more complicated than the
single site postulated in models of the MT channel pore
based on the properties of blockers (257). It then becomes
unclear which negative sites (in the vestibule or selectivity
filter; see FIGURE 10) might be crucial for binding Ca2⫹
and/or cationic blockers and whether these sites are the
same for the different antagonists. Significantly, many
blockers such as curare and DHS are polyvalent and may
interact with negative charges in both the vestibule and the
pore (23).
B. Location, Size, and Number of MT
Channels
A milestone in understanding hair cell mechanotransduction was the surprising proposal that the MT channel is
Physiol Rev • VOL 94 • JULY 2014 • www.prv.org
HAIR CELL MECHANOTRANSDUCER CHANNELS
A
B
Neutral residue
Negatively charged residue
–
Vestibule
Out
Out
–
K+
K+
K+
–
K+
–
–
K+
–
–
–
–
Ca2+
–
K+
Ca2+
Ca2+
–
–
K+
In
In
Low conductance
High conductance
C
Increasing conductance
D
Accessory
subunit
–
–
–
–
Out
K+
Channel
–
–
Ca2+
–
–
K+
In
E
Increasing conductance
FIGURE 10. Hypothetical structure of the MT channel illustrating the ion conduction pathway. A: crosssection of a channel showing a large vestibule on the external aspect of the channel and a tight selectivity filter
lined with negatively charged residues at the cytoplasmic end of the channel. It is proposed that in the
low-conductance isoform, there are critical neutral residues in the vestibule. B: in the high-conductance
isoform of the channel, these neutral residues are replaced with negatively charged residues that cause
accumulation of ions in the vestibule, hence increasing channel conductance (18). C: one scheme for
generating a range of single-channel conductances by mixing the low-conductance and high-conductance
isoforms in A and B in a tetrameric channel, assuming properties are a linear function of subunit composition.
D: in a second model, the MT channel comprises a pore-forming modulated by an accessory subunit with
negatively charged residues to generate a high-conductance isoform as in B. E: a scheme using the second
model for producing a range of unitary conductances, in which different numbers of accessory subunits, such
as LHFPL5 or TMC1, associate with the channel to systematically vary its properties (see sect. VIB).
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
localized to the tops of the stereocilia (107) and might be
activated by tension in the tip links (193). As argued by
those authors, the latter hypothesis could account for the
directional selectivity for bundle deflections towards its tallest edge, which would elevate tension in the tip links. The
hypothesis was pursued by specific experiments showing
that 1) hair bundle exposure to submicromolar extracellular Ca2⫹ severed the tip links and abolished MT currents
(12, 46, 73); 2) the channel blocker DHS was more immediately effective when iontophoresed near the top rather
than the bottom of the bundle (112); and 3) the first signs of
Ca2⫹ influx following bundle stimulation occurred principally at the tops of the stereocilia (54, 158). Defining the site
of Ca2⫹ influx through the Ca2⫹-permeable MT channels
was based on filling frog saccular hair cells with a fluorescent Ca2⫹ indicator and monitoring the initial bloom of
fluorescence with confocal or two-photon microscopy. This
approach was subsequently applied to rat cochlear hair cells
containing three stereociliary rows (19) and demonstrated
that the initial Ca2⫹ response was confined to the two
shorter rows, implying that the MT channels are present
only at the lower ends of the tip links. This conclusion
differed from the earlier supposition, where it was posited
that channels occurred at both ends of the tip link (54). The
more recent experiments (19) employed low-affinity Ca2⫹
indicators (Fluo4-FF) that would not be saturated by the
Ca2⫹ influx, and monitored the fluorescence with a fast (1
kHz) swept-field confocal that could follow Ca2⫹ diffusion
along the stereocilia. These two features helped distinguish
the Ca2⫹ signals at the tops and bottoms of the stereocilia.
The most significant factor though was the larger 0.5-␮m
diameter and more widely spaced stereocilia of IHCs compared with those in the frog bundle, making the signals
easier to resolve at the tips of the IHC stereocilia. The asymmetry in channel location accords with the asymmetrical
structure of the tip link, now known to be composed of
twisted pairs of cadherin-23 molecules at the upper end and
protocadherin-15 molecules at the lower end (124), and
would raise the possibility that each protocadherin-15
monomer interacts with one of the MT channels.
The extended extracellular domains of the two adhesion
molecules interact at their NH2 termini to form 150-nmlong filaments (73), the bond between them being stabilized
by Ca2⫹ ions (124, 232). The integrity of the tip links persists in endolymphatic Ca2⫹, but if the Ca2⫹ concentration
is reduced below 1 ␮M, buffered with EGTA or BAPTA, the
tip links are disrupted along with the transient ankle links
(12, 73, 84). Prolonged exposure to BAPTA also totally
abolishes mechanotransduction, but after brief (20 s) treatment, single-MT channel currents are visible in whole cell
recordings (18, 46, 129, 205; FIGURE 11); this provides a
method to quantify the channels without the impossibly
difficult task of cell-attached patch recordings from individual submicron stereocilia. Other approaches have found
channels in untreated cells in which there was virtually no
FIGURE 11. Experimental records of single MT channels. A: method of isolating single-channel events by
recording in whole cell configuration in which the majority of the tip links have been destroyed by exposure to
BAPTA. B: single-channel events in an apical rat IHC during imposition of a displacement step to the hair
bundle, the timing of which is given above. Four representative traces are shown illustrating evoked transitions
from closed (C) to open (O) state. The ensemble average of 12 stimuli is shown below displaying fast
adaptation. The amplitude histogram of the events indicates a single-channel current of 16 pA corresponding
to a conductance of 190 pS. C: single-channel events in an OHC from the middle turn of a rat cochlea, the
timing of the displacement step to the hair bundle being given above. The ensemble average also displays
adaptation, and the amplitude histogram of the events indicates a single-channel current of 11 pA, equivalent
to a conductance of 131 pS. In B and C, the holding potential was ⫺84 mV, and measurements were made
at room temperature. [B and C from Beurg et al. (18).]
972
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HAIR CELL MECHANOTRANSDUCER CHANNELS
Table 2. Single MT channel conductance
Hair Cell Preparation
Chicken vestibule
Frog saccule
Turtle auditory
Turtle auditory
Turtle auditory
Rat OHC
Rat IHC
Mouse OHC
Mouse OHC
Mouse OHC
Mouse IHC
Mouse IHC
Conductance, pS
Condition
50*
23†
110
30°C
Corrected to 25°C
2.8 mM Ca2⫹
80–160
150–300
100–144
180
112
80
63–110
90–320
67
2.8 mM Ca2⫹
0.05 mM Ca2⫹
Apex, middle
Apex, middle
Cochlear apex
Middle
Apex-base
0.05 mM Ca2⫹
1.5 mM Ca2⫹
References
Ohmori (183)
Holton and Hudspeth (101)
Crawford et al. (46)
Ricci et al. (205)
Ricci et al. (205)
Beurg et al. (18)
Beurg et al. (18)
Géléoc et al. (78)
Xiong et al. (271)
Kim et al. (129)
Pan et al. (187)
Beurg et al. (23); Caberlotto (28)
All measurements were made at a negative holding potential (approximately ⫺80 mV) in millimolar external
Ca2⫹ unless otherwise indicated, and at room temperature, except 30°C (*). †Conductance derived from
noise analysis at 10°C and extrapolated to 25°C using a Q10° ⫽ 1.45.
MT current (78, 183), or have drastically reduced the MT
current by treatment with the peptide channel blocker
GsMTx-4 (23). One method derived the amplitude from
analysis of current fluctuations (101). All are in agreement
that the MT channel has a large amplitude of 100 pS in the
absence of block by extracellular Ca2⫹ (cf., voltage-dependent K⫹ channels or acetylcholine receptor channels are
typically 30 pS or less), although unitary currents in vestibular hair cells are generally smaller than those in auditory
hair cells (TABLE 2).
Several conclusions can be drawn from the single-channel
recordings: 1) the single events can be driven with hair
bundle displacements of the same size and polarity as macrosocopic currents and can be blocked with DHS; 2) the
single-channel amplitude is increased about twofold when
extracellular Ca2⫹ concentration is switched from 1.5 mM
to an endolymphatic Ca2⫹; and 3) the single-channel open
probability determined from ensemble averages activates
rapidly and displays adaptation. These three observations
argue that the channels underlie the macroscopic MT currents (46, 129, 205; FIGURE 11D). A concern about the
magnitude of the channel conductance is that if two channels are connected to each tip link, then severing the tip
links with BAPTA should leave an even number of channels.
In the end just two channels should remain, and hence, the
conductance values may be twice that of a single-channel
event. For prolonged recordings, it is occasionally possible
to see multiple conductance levels, or with a fast recording
speed to see flickering between these levels. But the strongest evidence derives from the observation that using two
different methods to reduce the channel number, by BAPTA
or by peptide block, yield similar conductance values (23).
For channels isolated in this way, an unexpected finding
was that the unitary conductance varies along the auditory
epithelium, being smallest at the low frequency end. This
conclusion is true of all hair cells studied in turtle papilla
(205), but is restricted to OHCs in the mammalian cochlea
and no such tonotopic gradient is evident in IHCs (18, 23).
Increases in unitary conductance and in number of stereocilia per bundle means that there will be a severalfold gradient in the maximum MT current from low to high frequencies (see also FIGURE 8 for the chicken). Two questions
arise. First, what is the function of the gradient? Since there
will be greater Ca2⫹ influx in larger conductance channels,
this may account for the decrease in the fast adaptation time
constant, seen in both the macroscopic current and at the
single-channel level (205). A second question concerns the
underlying mechanism of the variation. The simplest explanation is that MT channels in OHCs at the two ends of the
cochlea are composed of different protein isoforms (FIGURE
10, A AND B) or have accessory subunits (FIGURE 10, D AND
E; see sect. VIB) that modify the channel conductance. One
specific hypothesis is that negative charges in the vestibule
of one isoform (FIGURE 10B) concentrate the cations and
increase the unitary conductance (18, 23).
With knowledge of the single-channel conductance and the
amplitude of the macroscopic MT current at a given location, a lower bound on the number of channels per tip link
can be derived. The maximum number of tip links was
obtained from stereociliary counts in scanning electron micrographs (18), and the number of channels per tip link was
calculated for turtle auditory hair cells (1.7, 205) and for rat
cochlear OHCs (1.8, 18) and IHCs (2.4, 19). Within measurement error, these values suggest there are two channels
per tip link, a surprisingly small number, but comprehensible if each channel makes direct connection to a component
of the extracellular tip link: each of the two channels could
connect to an intracellular domain of protocadherin-15 of
which there are two at the lower end of the tip link.
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
VI. THE MOLECULES OF THE
MECHANOTRANSDUCTION
MACHINERY
Myosin VIIa
Harmonin-b
Sans
A. Molecular Structure of the Transduction
Apparatus: The Tip-Link Complex
The main experimental approach that has driven the search
for the molecular underpinning of hair cell mechanotransduction over the last 15 years has been the exploration of
inherited forms of deafness (210), and to date, close to 100
mutations have been identified (http://hereditaryhearingloss.org). The majority is linked solely to inner ear defects and
is known as nonsyndromic, denoted either as DFNBn (autosomal recessive) or DFNAn (autosomal dominant). The
largest syndromic group comprises those of Usher syndrome (210, 230), types I, II, and III, named for Charles
Usher, who was one of the first to examine transmission of
this recessive deafness and blindness disorder. Type I is the
most severe and is linked to profound deafness at birth
attributable to hair cell degeneration combined with progressive photoreceptor degeneration that begins during the
first decade of life (230). Remarkably, five of the proteins
mutated in Usher type I form part of the upper tip-link
density (myosin VIIa, harmonin, and sans; Ref. 85) or the
tip link itself (cadherin-23 and protocadherin-15; Ref. 124).
This set of proteins also occurs in the calyceal microvilli
linking the photoreceptor inner and outer segments in primates but not in mice (221), which may structurally reinforce the cilial link between inner and outer segments.
In mammalian cochlear hair cells, cadherin-23, the upper
component of the tip link, is thought to be attached to the
stereociliary actin cytoskeleton through its intracellular
COOH terminus associating with harmonin-b, a PDZ-domain protein, and with sans and myosin VIIa (FIGURE 12;
Ref. 85). Quantitative immunofluorescence was used to
demonstrate that the ternary complex clusters at the upper
tip-link density with an approximate stoichiometry of three
harmonin-b molecules to one each of the two others. The
simplest hypothesis is that myosin VIIa is the motor that
maintains tension in the tip link (14, 85). There is no evidence in mammalian OHCs that this function is usurped by
myosin Ic, as has been proposed for vestibular hair cells
(100, 234). In support of its localization at the UTLD, mutations in harmonin result in a loss in sensitivity and slowing of adaptation in the MT currents (87, 173), although
the effects are more subtle than might be expected.
One of the more conspicuous achievements in recent haircell biology has been the structural identification of the
tip-link complex. Cadherins are normally regarded as mediating cell-cell interactions, but in the tip link they join one
part of the cell with another by means of large extracellular
domains. A combination of evidence, including the absence
of tip links in cadherin-23 (USH1D) mutants, immunolo-
974
USH1B
USH1C
USH1G
Cadherin-23 USH1D
Protocadherin-15 USH1F
K+, Ca2+
LHFPL5
MT channel
Myosin XVa
Whirlin USH2D
CIB2
USH1J
Eps8
Actin
Top connectors
(stereocilin)
FIGURE 12. Schematic of the tip link and its attachments at the
top of a shorter stereocilium and at the side wall of its taller neighbor. The prevailing view of the molecular composition of the transduction complex (see text) is illustrated, including the tip link (124),
the upper tip-link densities (tripartite complex of myosin-7a, harmonin-b, and sans; Ref. 85) and the lower tip-link densities (myosin
XVa, whirlin, and Eps8; Refs. 53, 16, 161) and CIB2 (Ca2⫹ and
integrin binding protein isoform 2; Ref. 201). Many of the proteins
are mutated in Usher syndrome. In addition, the MT channel is
localized to the lower end of the tip link (19), with LHFPL5 (also
known as TMHS, Ref. 271) as a possible coupling protein.
calization of the protein to near the tops of the stereocilia,
and the consistency of the size of the extracellular portion of
the protein with the ⬃150-␮m tip-link length led to the
conclusion that cadherin-23 was a component of the tip link
(229, 231). Similar arguments were made for protocadherin-15 (USH1F; Ref. 2), culminating in the proposal that
the tip link is a heterophilic complex of homodimers of
cadherin-23 at its upper end and homodimers of protocadherin-15 at its lower end (124) joined at their NH2 termini.
The extracellular region of each cadherin is assembled from
multiple ectodomains (EC): 27 EC repeats for cadherin-23
and 11 EC repeats for the shorter protocadherin-15. In addition to binding to the EC subregions of each cadherin, Ca2⫹ is
required for formation of the heterophilic complex; consequently, removal of extracellular Ca2⫹ is expected to sever the
tip links as observed experimentally. Evidence for a functional
interaction between cadherin-23 and protocadherin-15 was
provided by showing that regeneration of tip links severed by
low Ca2⫹ could be blocked by extracellular fragments of either protein (150). This scheme places the two strands of protocadherin-15 at the lower end of the tip link where they might
convey force (directly or indirectly) to a pair of MT channels
located at the tops of the stereocilia (19).
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HAIR CELL MECHANOTRANSDUCER CHANNELS
Thus protocadherin-15 becomes a central player in the
transduction apparatus and might interact with the MT
channel. It occurs in three isoform classes, differing in their
cytoplasmic domains CD1, CD2, and CD3 (2, 267). Absence of protocadherin-15-CD1 or -CD3 had no effect on
hair-bundle morphology or hearing, but mice lacking protocadherin CD2 were deaf (267). The most prominent abnormality was loss of links between the kinocilium and the
tallest stereocilia and an associated abnormal polarization
of the bundles, suggesting that the protocadherin-15-CD2
isoform is localized to the kinocilium and influences bundle
orientation orchestrated by the planar cell polarity proteins.
The kinociliary links, like the tip links, are oriented along
the bundle’s axis of symmetry and are also composed of
cadherin-23 and protocadherin-15, but their polarization is
opposite to that of the normal tip links, with protocadherin-15 being at the kinociliary extremity of the link and
cadherin-23 facing the stereocilium (83, 267). Under some
cirumstances, functional interciliary links can also be made
from homophilic complexes of PCDH15. After tip-link destruction with BAPTA, the links regenerate at the tops of
the stereocilia along with recovery of mechanotransduction
over a period of 24 h (111, 275). During the initial phase of
regeneration, shorter tip links comprising only PCDH15
appear first but are still able to mediate MT currents of
normal amplitude (111). Only later do the mature hetereophilic CDH23/PCDH15 links materialize.
Usher syndrome has so far shed little light on the identity of
the MT channel or of the composition of the lower tip-link
density. The genes responsible for two Usher type I subtypes
are unknown, and the gene for USH1J was recently identified as a Ca2⫹- and integrin-binding protein (CIB2) present
in the stereocilia (201). A type II gene, USH2D, encodes
whirlin, a PDZ protein present at the tops of the stereocilia
that interacts with the myosin XVa (16, 53), also localized
to the stereociliary tips, along with the actin-regulatory protein Eps8 (161). The three proteins, myosin XVa, whirlin,
and Eps8, are thus proposed as components of a complex at
the stereociliary tips and may be a major contributor to the
lower tip-link density. Other proteins at the tips of the stereocilia that may enhance the lower tip-link density include
Eps8L2 (74), twinfilin-2 (191), myosin IIIa (224), and espin
1, a long isoform of espin containing an NH2-terminal
ankyrin repeat domain (222). The protein complex may
have two important and interrelated roles: anchoring the
MT channel to the actin cytoskeleton and regulating actin
polymerization and elongation of the stereocilia actin core,
thus being a major factor determining the stereociliary
height and the staircase. For example, Eps8 and Eps8L2
collaborate to maintain the hair bundle staircase structure,
which is lost in their absence (74); myosin IIIa transports
espin 1 to the tips of the stereocilia and causes their elongation (222). In contrast, twinfilin-2 has an opposite action to
reduce stereociliary length (191). The actin bundling protein fascin-2 is also concentrated towards the tips of the
bundle’s longest stereocilia; its expression increases during
stereociliary elongation, and its mutation affects bundle
height (226). Clearly, a large and disparate set of proteins is
involved in regulating stereociliary length and staircase.
The lower tip-link density bears comparison with the postsynaptic density on dendritic spines (125), both of which
may harbor multimolecular complexes associated with signal transduction. Synaptic transmission at the tip of the
spine relies on AMPA receptors, ligand-gated ion channels
opened by the binding of glutamate neurotransmitter. Mechanical transduction is mediated by MT channels at the
tips of the stereocilia, which are of similar size and shape to
the dendritic spines. In the two structures, Ca2⫹ signaling
and homeostasis play a crucial role in functional maintenance, although stereocilia possess an actin-based internal
core different from that of the spine. Nevertheless, in both
cases, the transduction complex may have equivalent accessory proteins to regulate the insertion, localization, and
properties of the ion channel. Examples are the transmembrane AMPA-receptor regulatory proteins (TARP) family
of proteins in the dendritic spine which influence the expression, kinetics, and conductance of the AMPA receptors
(239). A tetraspan protein similar to the TARPs is TMHS
(tetraspan membrane protein of hair cell stereocilia, elsewhere referred to as LHFPL5), a four transmembrane-domain protein that exists in the stereocilia of IHCs and
OHCs, and its gene-mutation underlies deafness in mice
(155) and humans (225). LHFPL5 has been proposed to
link PCDH15 to the channel complex, and its knockout or
mutation in the hurry-scurry (hscy) mouse affects integrity
of the tip link and also, significantly, reduces the MT channel unitary conductance (271).
B. Molecular Structure of the Transduction
Apparatus: The Ion Channel
A number of channel-protein candidates have been advanced as being components of the MT-channel pore, but
none has so far survived careful scrutiny (262). Several criteria must be satisfied to offer definitive evidence in support
of a given candidate: 1) it must be present in the hair bundle
as demonstrated by immunolabeling and/or exogenous expression of a GFP-labeled transcript; 2) mutation or knockout of the candidate gene should be associated with the
absence of transduction and behavioral deafness, and the
defect should be rescuable by exogenous introduction of
the normal transcript; 3) expression of the protein in a heterologous cell system, such as HEK293 cells, should bestow a
new ion channel with the appropriate pore properties (ion
selectivity, blockers and unitary conductance), preferably
gated by mechanical stimulation. Unfortunately, there are
confounding circumstances for each of these criteria, including the nonspecificity of antibodies, the presence of
multiple isoforms that can substitute for each other (e.g.,
TRPC channels, TMCs), and the requirement for other ac-
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
cessory proteins to secure heterologous expression. Problems with immunolabeling might arise because there are
likely to be only a few channel proteins in the stereocilia and
also the hair bundle glycocalyx is “sticky” causing nonspecific binding. Therefore, antibody specificity must be verified by its absence in the appropriate knockout. A good
example is the antibody to the NH2 terminus of the nonselective cation channel HCN1, which labeled hair bundles
suggesting it could be a stereociliary channel; however, this
antibody also decorated hair bundles from HCN1 null
mice, demonstrating the labeling was an artifact (102). The
channel protein might have been missed because it is essential elsewhere in the body and its gene-knockout is embryonically lethal.
TRP channels, named for the transient receptor potential
phenotype in Drosophila photoreceptors, are six transmembrane-domain proteins creating tetrameric channels
redolent of voltage-gated K⫹ channels. Several members of
the TRP channel superfamily have been put forward as
candidates for the MT channel, based on observations that
this heterogeneous family includes members with unusual
mechanisms of activation including mechanoreception
(189). This is exemplified by TRPN (NOMPC) which, on
account of the reduction in the receptor current in the
nompC mutant, was proposed as a component of the
mechanotransduction apparatus in Drosophila bristle organs (266). TRPN is crucial for gentle touch in Drosophila
larvae, and expression of TRPN in a heterologous system
has shown that it forms a mechanosensitive cation channel
(272). However, nompC is absent from the mammalian
genome. Other members of the vertebrate TRP superfamily
have been considered including TRPA1, TRPML3, and one
or more isoform of TRPC. Immunolabeling localized the
TRPA1 protein to the hair bundle, especially the stereociliary tips, and such labeling was lost when transduction was
disrupted by severing of the tip links; furthermore, knockdown of the transcript attenuated the MT current (39).
When expressed heterologously, it produced an ion channel
with properties comparable to the MT channel (177), but
with no evidence for mechanosensitivity. However, it was
later discovered that Trpa1 null mice exhibited no defects in
audition or balance (144). Moreover, immunolabeling of
the hair bundle with the TRPA1 antibody persisted in the
knockout, indicating it was an artifact. Why knockdown of
Trpa1 reduced the MT current is still unclear. TRPA1 is
most highly expressed in dorsal root ganglion nociceptors
and may mediate sensations such as cold and itch (144).
The evidence for TRPML3, encoded by the mucolipin-3
gene, is more complicated. It is expressed in a number of
tissues including the inner ear, and its gene mutation in the
varitint-waddler mouse shows deafness and balance defects
along with other problems (13). In cochlear hair cells of
mutants, the hair bundles become disorganized and the
MT currents are reduced (but not lost). The normal pro-
976
tein is localized to the hair cells including at the base of
the hair bundle, but the latter localization is lost in the
varitint-waddler (255). Recording in hair cells and in
heterologous systems have demonstrated that TRPML3
forms an inwardly rectifying cation channel permeable to
Ca2⫹, and the point mutation in the varitint-waddler
renders it constitutively active, thus loading the cells with
Ca2⫹, a byproduct thought to lead to hair cell degeneration (88, 178, 255). More recent work has shown that the
TRPML3 channel is also localized to the endosomes and
lysosomes, and because channel opening is pH sensitive,
it may regulate the acidification of early endosomes and
be important in the endocytic pathway (13, 88). As a
postscript to this story, targeted knockout of the
TRPML3 gene surprisingly displayed no auditory or vestibular defects (120).
Unlike the other two families, there was prior evidence for a
role of the canonical transient receptor potential family
TRPC in mechanotransduction, especially TRPC6 in the
muscle membrane (189). Moreover, the founder member of
the TRP superfamily, the transduction channels in Drosophila photoreceptors (to which the vertebrate TRPC
channels are most closely related), were recently suggested
to be mechanosensitive (95). However, there is debate over
whether TRPC6, or other TRPC channels, is directly gated
by force rather than indirectly via a G protein-coupled pathway (189). Trpc3 and Trpc6 transcripts are present in cochlear hair cells (197), and double knockouts of Trpc3 and
Trpc6 (but neither alone) produced a moderate high-frequency hearing loss and reduction of MT currents in basal
OHCs (197). TRPC3 immunolabeling occurred on the hair
cell membrane, and the channels could be activated by depletion of cytoplasmic Ca2⫹, but a G protein-coupled receptor pathway was implicated (198). It seems unlikely that
these proteins form the MT channel, although they may be
involved in hair-cell Ca2⫹ homeostasis, disruption of which
almost certainly impairs mechanotransduction. Confinement of the effects of knockouts to basal high-frequency
OHCs may reflect the higher metabolic vulnerability of
basal OHCs and their susceptibility to Ca2⫹ loading (20).
These three TRP-channel examples emphasize how in
searching for the hair-cell MT-channel protein, incomplete
evidence can be deceptive and lead to false conclusions.
Indeed, for all three TRP channels, knocking out the genes
failed to produce profound deafness or complete abolition
of MT currents.
The strongest evidence currently available is for a member
of the transmembrane channel-like (TMC) protein family
(123). TMC isoform-1, the founding member of the family,
is a ⬃760-amino acid protein predicted to contain multiple
(6 or more) transmembrane domains (145). In contrast to
those TRP channels described above, knockout of Tmc1
causes profound deafness (142, 236) due to complete loss of
MT currents in mice after the first postnatal week (130).
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HAIR CELL MECHANOTRANSDUCER CHANNELS
The TMC family has eight genes (143, 176), of which at
least two (Tmc1 and Tmc2) occur in the mammalian cochlea (123) and four (TMC1, TMC2, TMC3 and TMC6) in
the chicken basilar papilla (176). The dn and Beethoven
mutations of the Tmc1 gene in mice are associated with
hearing loss, absence of cochlear microphonics, and
OHC degeneration (142, 162, 236, 263). There are also
equivalent human nonsyndromic deafnesses, an autosomal dominant deafness, DFNA36, that is progressive
and a recessive profound deafness DFNB7/11 linked to
TMC1 mutations (142); there are over 30 mutations of
the TMC1 gene associated with human deafness making
it one of the more common nonsyndromic deafnesses
(97). However, it has been unclear whether deafness
stems from lack of MT channel function or is secondary
to a developmental defect (162). Nevertheless, double
knockouts of Tmc1 and Tmc2 were reported to abolish
MT currents in mouse auditory and vestibular epithelia,
a defect that could be rescued by transfection with either
Tmc1 or Tmc2 message (123). Thus TMC1 satisfies the
second criterion. While these results are suggestive, there
are inconsistencies about whether the MT current vanishes in Tmc1 mutants. Complications arise because of
the developmental changes that occur during the first two
neonatal weeks prior to the onset of hearing in mice
(162). For example, over this period there are changes in
cellular morphology and ion channel complement and
also downregulation of Tmc2 in the cochlea (123).
Although mRNA for Tmc1 and Tmc2 is found in cochlear
and vestibular hair cells (123), the presence of the protein in
the stereocilia is uncertain. For example, antibody labeling
indicated that in avian hair cells, TMC2 protein was principally associated with the lateral membranes (176). In contrast, transfection of mouse hair cells with Tmc2-GFP
showed localization to the tops of the stereocilia (123).
With neither labeling technique is there evidence on the
subcellular localization of TMC1 in mammals. Thus they
do not fully satisfy the first criterion. Attempts to express
TMCs in heterologous systems have also been ambiguous.
When TMC1 from C. elegans was expressed in several
mammalian cell lines, it generated a cation conductance
that was activated by high extracellular Na⫹, although the
channel was not mechanically sensitive (31); however, this
result has not been replicated for vertebrate isoforms, and
there is again no evidence for mechanosensitivity, thus not
fulfilling the third criterion. Circumstantial support for a
role in the ion conduction pathway has emerged from
the observation that, in Tmc1 or Tmc2 gene knockouts, the
Ca2⫹ permeability and the unitary conductance of the
MT channel were altered (129, 130, 187). Taken at face
value, these results support a role for the TMC proteins in
forming the pore, a conclusion strongly reinforced by the
finding that a point mutation, M412K, in theTmc1
Beethoven mutant conferred a reduced single MT channel
conductance and Ca2⫹ permeability (187).
However, the waters have been muddied by the discovery
that mechanically sensitive currents can still be evoked by
bundle displacements in mice with mutations in both Tmc1
and Tmc2 (23, 129). The current in the double mutant was
blocked by MT channel blockers, such as DHS and
amiloride, but it was anomalous in being activated by large
bundle deflections away from its tallest edge, and therefore
opposite in stimulus polarity to that in the wild type. It
should be noted that these anomalous currents were recorded with similar properties in Tmc1dn/dn Tmc2⫺/⫺
and in Tmc1⌬/⌬ Tmc2⫺/⫺, which are derived from two
different Tmc1 mutants (23), implying they are not products of still functional (but truncated) TMC protein. MT
currents responding to the opposite stimulus polarity have
also been recorded in certain Pcdh15 and Cdh23 mutants in
which the tip links are lost (3). In line with this finding, the
MT current in the Tmc1/Tmc2 double mutants was unaffected by treatment with BAPTA to break the tip links,
implying that the channels were not activated in a conventional manner by increased tip-link tension (129). Where
the channels underlying the anomalous current are located
in the bundle, and why they respond to reversed polarity
displacements of the bundle remain to be determined. Nevertheless, their existence argues that the mechanosensitive
current in the Tmc1/Tmc2 double mutants may flow
through the MT channels, but these channels are mislocalized and not attached to the tip links. A corollary is that the
TMC proteins are not pore-forming subunits of the MT
channel but more likely accessory and/or trafficking proteins. Lack of correct targeting to the stereociliary tips, so
preventing interaction with other components of the transduction complex (e.g., LHFPL5; Ref. 271), may account for
the altered pore properties.
Thus the attributes of the TMC proteins resemble those of
auxiliary proteins for other ion channels, for example, the
TARP proteins (tetraspan membrane proteins weakly homologous to LHFPL5) and the cornichon (CNIH) proteins.
The TARP and CNIH proteins may act synergistically to
influence expression and gating properties of AMPA receptors at glutamatergic synapses on dendritic spines (239).
For example, CNIH-2 association increases both the Ca2⫹
permeability and single-channel conductance of GluA1 receptors (37). It is generally thought that besides controlling
membrane expression of the GluA1 channel, both TARP
and CNIH proteins bind to the channel to modulate its
properties. By analogy, the simplest hypothesis for the hair
cell proteins is that LHFPL5 and TMC1 (and possibly
TMC2) bind to the MT channel and in their presence, the
channel conductance is increased. The implication is that
the tonotopic gradient in MT channel conductance is conferred by TMC1 and possibly other subunits and does not
stem directly from different isoforms of the pore-forming
subunit (see FIGURE 10, C AND E). Thus, in both the Tmc1
mutant and the Tmc1/Tmc2 double mutant, the singlechannel conductance is similar at the apex and base (FIGURE 13;
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ROBERT FETTIPLACE AND KYUNGHEE X. KIM
FIGURE 13. Single mechantransducer channels in mouse cochlear hair cells. A: examples of single-channel
currents recorded in an apical OHC for step hair-bundle deflections, stimulus monitor shown above; closed (C)
and open (O) states are indicated beside traces. B: amplitude histogram of events like those in A, giving
single-channel current of ⫺5.2 pA at ⫺84 mV holding potetnial. C: single-channel currents in a basal OHC for
step hair-bundle deflections. D: amplitude histogram of events like those in C, giving single-channel current of
⫺8.5 pA at ⫺84 mV holding potetnial. E: mean single-channel currents (left axis) and conductance (right axis) ⫾
SD, plotted against cochlear location, expressed as the fraction of the distance along the basilar membrane
from the low-frequency apical end. The current is larger at the base for wild-type OHC but not for Tmc1⫺/⫺
OHC in which the tonotopic gradient is virtually abolished. Number of cells: wild-type OHC apex, 6; middle,
5; base, 6; OHC from deafness mutant, Tmc1dn/dn: apex 17, middle 3, base 7. Mouse age range was
P2 to P6.
Ref. 129). Definitive evidence on the nature of the interaction awaits clarification of the channel’s molecular identity.
However, the current conjecture is that both TMC1 and
LHFPL5 behave like auxiliary subunits for the MT channel
and are able to alter its permeation properties. A third membrane protein, that has been studied less but may have a
parallel role, is TMIE, the mutation of which is associated
with deafness in humans and mice (174), and loss of transduction in zebrafish (79).
C. The Mechanism of MT Channel Activation
and the Role of the Membrane Lipids
The first mechanotransducer channel to be identified was
the bacterial MscL channel, and its mechanical sensitivity
was revealed after reconstituting the purified channel protein into a lipid bilayer (141). Such bilayers consisted of
defined lipids and contained no other proteins, the unavoidable conclusion being that the MscL channel was mechanically sensitive by directly detecting the tension in the bilayer. The MscL channel is a homopentamer of 136 residue
subunits, each forming two transmembrane domains (30).
On the basis of X-ray diffraction studies, stretch of the
bilayer membrane is thought to pull the transmembrane
domains outwards and tilt them, so the MscL barrel expands and adopts a flatter conformation (237); the central
978
pore region has been proposed to open like an iris, thereby
creating a nanometer-sized channel having an ionic conductance of 3 nS. On this type of mechanism, it is natural to
suppose that channel activation might be sensitive to the
lipid composition of the bilayer. Possible factors include
lipids that alter the thickness and fluidity of the membrane,
which could affect the transition rate constants and stability
of the open conformation, as well as the surface charge and
membrane curvature (195, 278). Given these influences,
stability may be accomplished by specific lipids congregating around the ion channel to optimize its function. Prime
examples of this compartmentalization are the lipid domains consisting of cholesterol and sphingolipids that assemble with proteins into platforms tens of nanometers
wide (154). Such platforms might be anchored to the cytoskeleton and act as a mechanism for focusing force stimuli
onto mechanosensitive channels embedded in the lipid raft
(5). Since the lipid platform itself could be attached to the
cytoskeleton, this arrangement does not explicitly require
the MT channel also to be anchored to the cytoskeleton.
There is evidence for lipid specialization towards the top of
the hair bundle. Phosphatidylinositol 4,5-bisphosphate
(PIP2) is enriched in the top half of the stereocilia, and PIP2
depletion with phenylarsine oxide or quercetin has been
shown to reduce the amplitude and adaptation of MT cur-
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HAIR CELL MECHANOTRANSDUCER CHANNELS
rents in frog vestibular hair cells (99). Another membrane
lipid, cholesterol, detected by the fluorescence of the cholesterol-binding antibiotic filipin, may be localized at the
very tips of the stereocilia (274), although an earlier freezefracture study did not find any filipin-induced deformations
at the stereociliary tips (69). In the latter study, the membrane at the tip of the stereocilium exhibited a number of
large particles of ⬃11.0 nm in diameter that could conceivably correspond to the MT channels. So what is the proximate stimulus for MT channel activation? Is it force between the extracellular and intracellular channel connections to the tip link and the cytoskeleton, respectively, or
does the hair cell MT channel, like MscL, detect changes in
the bilayer tension (36, 141)? These extremes are depicted
in FIGURE 3. A related question pertains to the physical
manifestation of the gating spring and the origin of its stiffness; this gating spring has been hypothesized as a means of
coupling bundle displacement with force delivery to the MT
channel (see sect. IIIA). The most obvious manifestation of
the gating spring is the tip link itself, but calculations based
on its structure have suggested that it is too stiff to account
for the ⬃1 mN/m associated with channel activation (121,
232). A second site is in the channel attachments to the
cytoskeleton or to the surrounding membrane. The tip link
is probably attached directly or indirectly to the MT channel. The stoichiometry of two PCDH15 molecules and a
pair of MT channels is circumstantial evidence for mechanical coupling; furthermore, LHFPL5 might be a coupling
protein since it regulates expression of PCDH15, and likely
interacts with the channel whose ion conduction properties
it can influence (271). The problem has been further addressed by modeling the extent to which the mechanical
properties of membrane lipids can explain channel activation and kinetics (194). Indirect evidence supporting the
involvement of membrane stretch is that in transmission
electron micrographs, the plasma membrane at the top of
the stereocilia, where the tip link inserts, is pulled away
from the LTLD to produce a tentlike appearance (FIGURE
3A). The modeling concluded that the properties of the bilayer are sufficient to explain channel activation in frogs,
but because of the larger gating stiffness in mammals, a
cytoskeletal tether may also be required. However, the
modeling did not consider the possibility that the membrane stiffness might be augmented by the presence of cholesterol in an organized membrane platform. This will be
resolved only when the full protein composition of the
channel and the LTLD has been established.
a nonlinearity in the hair bundle mechanics, attributable to
force generated during channel gating. Consequently, channel reclosure during Ca2⫹-dependent fast adaptation was
proposed to generate a force to oppose the stimulus, and
this was taken as evidence that the bundle can perform
work. Since the speed of adaptation changes along the cochlea, force generation is high-pass filtered with a corner
frequency that may be matched to the CF of the appropriate
cochlear location. However, further progress in elucidating
the mechanism has been partly hindered by the inability to
isolate the channel protein, and it was not until the advent
of molecular genetics that the complexity of the transduction complex became apparent. Knowledge of the composition of the tip link, a helical dimer of CDH23 and
PCDH15, and the protein complex at the upper attachment
point including myosin VIIa, gleaned largely from studies of
Usher syndrome, provided a starting point. Localization of
the MT channel to the lower end of the tip link facilitates an
unbiased search for the channel itself and its protein partners which might interact with PCDH15. Recent results
from mouse mutants have suggested that the tonotopic variation in single MT-channel conductance may be at least
partly due to gradients in accessory subunits such as LHFPL5
and TMC1, but more work is needed to clarify the basis of
channel diversity. There are several unresolved questions
about the MT channel function including the location of the
Ca2⫹-binding site for fast adaptation and the mechanism of
channel activation, whether it senses force delivered
through accessory proteins or is gated indirectly by deformation of the lipid bilayer. In the latter case, what part does
the lipid environment play in gating? It will also be important to provide definite evidence one way or the other on
whether hair bundle motilty makes any contribution to amplification and tuning of the mammalian cochlea. More
thorny questions pertain to how the channels are targeted to
the stereociliary tips and how incorporation into the transduction complex and attachment to the tip links is regulated. It is hoped that the channel will be identified in the
near future, which should enable many of these questions to
be addressed in a rational manner.
ACKNOWLEDGMENTS
We thank Maryline Beurg for helpful comments, Carole
Hackney for providing electron micrographs, and the anonymous reviewers for their many helpful comments.
Address for reprint requests and other correspondence: R.
Fettiplace, 185 Medical Sciences Bldg., 1300 University
Ave., Madison, WI 53706 (e-mail: fettiplace@wisc.edu).
VII. CONCLUSIONS
Over the period during which hair cells have been studied
physiologically, substantial progress was initially achieved
in defining the characteristics of hair-cell mechanotransduction, including its force sensitivity, kinetics, Ca2⫹-dependent adaptation, ionic selectivity, and single-channel conductance. An important insight stemmed from discovering
GRANTS
Work in the authors’ lab was supported by National Institute on Deafness and Other Communication Disorders
Grant RO1 DC01362 (to R. Fettiplace).
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DISCLOSURES
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