Selective Permeability
Jackie Bonds
January 23, 2012
Neus 586
Monday, January 30, 2012
Selective Permeability – What
• Selective permeability means that the
cell membrane has some control over
what can cross it, so that only certain
molecules either enter or leave the cell. Monday, January 30, 2012
Regulation of Membrane
Permeability
• Ionic composition inside neurons is
tightly regulated and must be able to
be rapidly modified in response to
various stimuli
• Specialized transmembrane channels
allow ions to rapidly enter the cell
while still maintaining high selelctivity
• Voltage-gated channels
• Ligand-gated channels
Monday, January 30, 2012
Neural Signaling
• Resting potential is the
result of an equilibrium
between the electrical
and chemical forces
acting upon Na+ and K+
• Depolarization is result
of a stimulus large
enough to cause voltagegated Na+ channels to
open
• Repolarization is the
Monday, January 30, 2012
What properties govern the
specificity of channels? How
Monday, January 30, 2012
Techniques
•
•
•
•
X-ray crystallography
Electron Density Maps
Pharmacology
Genetics
– Sequencing
– Site-directed mutagenesis
• Patch-clamping
Monday, January 30, 2012
Voltage-Gated Ion Channels
• Voltage-gated ion channels are like the
transistors of logical circuits, detecting,
amplifying, and reshaping electrical
messages
• Hodgkin and Huxley succeeded in
describing the permeability changes as a
set of chemical reactions whose rate
constants are a function of voltage
“…changes in ionic permeability depend on
the movement of some component of the
membrane which behaves as though it had
a large charge or dipole movement…to
Monday, January 30, 2012
Visualization of the channel
• Structure and function has been studied
using X-ray crystallographic studies
• Elucidated an important feature of the
selectivity filter  to create a queue of
cation binding sites that mimic the waters
of hydration surrounding that specific
cation
• Allows the observation of the channel
structure in various chemical
environments
• Allowed the visualization of ion position
Monday, January 30, 2012
General Features of a VGIC
SF = Selectivity Filter
VS = Voltage Sensor
OV = Outer Vestibule
IV = Inner Vestibule
Monday, January 30, 2012
Armstrong et al.
1998
Voltage Sensor (S4 Segment)
• Cysteine mutagenesis and cysteine labeling
with thiol-reactive compounds has provided
good evidence that the S4 segments in both
Na+ and K+ channels move as expected
following voltage changes
• State dependent labeling  a specific cysteine
may be labeled only from the outside when
the channel is open and only from the inside
when the channel was closed. The labeling
patterns are consistent with the outward
movement of S4 (the voltage sensor) in
Monday, January 30, 2012
Potassium Channel
• Potassium conduction
through their specific
channels is critical for
cell volume regulation,
hormone secretion, and
electrical impulse
formation (neural
signaling)
• Signature Sequence
forms the selectivity
filter which prevents the
passage of sodium ions
but allows potassium
ions to conduct across
the membrane at rates
approaching the
Monday, January 30, 2012
MacKinnon.
2003
Channel Architecture
• Since the inner helices are
tilted slightly inward and
kinked with respect to the
membrane, the subunits
open like the petals of a
flower facing the outside of
the cell. Gives the
appearance of an inverted
teepee
• This architecture is likely a
general feature of cation
channels with four inner
helices, four pore helices,
and a selectivity filter each
tuned to select the specific
ion at the extracellular
Monday, January 30, 2012
MacKinnon et al. 1998
• The channel pore is
made up of four
subunits, each of
which contain two
fully transmembrane
α-helices (an inner
and outer) and a tilted
pore helix that runs
half-way through the
membrane and points
its negative end
Monday, January 30, 2012
MacKinnon et al. 1998
Ion Selectivity and Movement
• A K+ ion could move through the internal
pore and cavity without shedding its waters
of hydration but the selectivity pore is so
narrow that it must shed its hydrating waters
to enter. The ion is stabilized in the
selectivity pore because it is lined exclusively
with polar main chain atoms found in the
signature sequence.
• The configuration of these ions within the
selectivity pore allows the channel to exploit
the electrostatic repulsive forces to
Monday, January 30, 2012
• As the ion encounters
the selectivity filter, it
interacts with 4 evenly
spaced layers of carbonyl
oxygen atoms and a
single layer of threonine
hydroxyl oxygen atoms
 this creates 4 binding
sites where K+ binds in a
dehydrated state and is
surrounded by 8
oxygens that essentially
mimic the arrangement
of water molecules
around a hydrated ion
Monday, January 30, 2012
MacKinnon. 200
Visualization of Ion Position
• All K+ channels show a
selectivity sequence of K+ ≈
Rb+ > Cs+. Permeability for
the smaller ions such as Na+
and Li+ are immeasurably
low
• Rb+ (1.48Å) is almost a
perfect analog to K+ (1.33Å)
due to the similarity in size
and permeability
characteristics. Rb+ (as well
as Cs+) is more electron
dense than K+ making it
very suitable for use in
visualizing the locations of
Monday, January 30, 2012
MacKinnon et al. 199
Isolating the Channel
• All K+ channels can be blocked by
tetraethylammonium (TEA)
• TEA interacts with the amino acids that
define the entryway into the pore and blocks
the K+ channel at both sides of the
membrane at distinct sites. The amino acids
that TEA cations interact with are located
just external to and internal to the structure
that forms the selectivity filter. TEA cannot
enter the selectivity filter, only block it.
Monday, January 30, 2012
Functional Experiments
• The transmembrane voltage is
imposed across the relatively short
distance through the selectivity filter
which is ~12Å (Length of pore = 45Å)
 the rate-limiting steps for a K+ to
travel through the channel occur in the
short span of the selectivity filter
• TEA ions can only traverse ~20% of the
transmembrane voltage
Monday, January 30, 2012
Summary of K+ Channel
• The pore is constructed like an inverted teepee, with the
selectivity filter held at its wide end. This is most likely
applicable to other cation channels such as Ca2+ and Na+
• The narrow selectivity filter is ~12Å long and has a polar lining,
whereas the remainder of the pore is wider and has a relatively
inert hydrophobic lining. This favors high K+ throughput by
minimizing the distance over which K+ interacts strongly with
the channel
• The large water filled cavity and helix dipoles help to overcome
the high electrostatic energy barrier in the low dielectric
membrane center
• The K+ selectivity filter is lined with carbonyl oxygen atoms
which provide 4 closely spaced binding sites and is constrained
to an optimal geometry that will only fit a dehydrated K+ ion
• Two K+ ions in close proximity within the filter exude repulsive
forces on each other, which helps to overcome the otherwise
Monday, January 30, 2012
Monday, January 30, 2012
Inactivation
• Inactivation gating is a second gating
factor that is mechanistically simpler
and different from activation  after
the activation gate opens, a portion of
the channel peptide diffuses into the
mouth of the inner vestibule of the
pore and blocks conduction (a foot in
the door sort of mechanism)
Monday, January 30, 2012
Inactivation (cont.)
• Site-directed mutagenesis by Aldrich
et al. found that deletions within the
first 20 amino acids of the N-terminus
completely destroy fast inactivation.
Even more fascinating was the finding
that deletions in the next 63 amino
acids resulted in speeding inactivation
– Cutting off the inactivation particle with
pronase removed the inactivation ability
Monday, January 30, 2012
CNS Pathologies
• Many of the ion channel diseases are
known as paroxysmal disorders in
which the principal symptoms occur
intermittently in some individuals who
otherwise may be healthy and active
Monday, January 30, 2012
Epilepsy
• Epileptic seizures are behavioral
attacks resulting from the overly
synchronized and excessive activity of
large groups of neurons.
– Symptoms: Alterations/loss of
consciousness, sustained or rhythmic
muscle contraction, stereotyped gestural
movements, and visual/somatosensory
hallucinations
Monday, January 30, 2012
Epilepsy (cont.)
• The SCN1B (the β1 auxiliary subunit of
the voltage gated Na+ channel) genes
of epileptic individuals contained a
single base-pair substitution which
caused a single amino acid change in
the protein sequence
– The β1 subunit is important for the timing
of channel open and close
– A mutation in this region results in a loss
of function  cells expressing the mutant
Monday, January 30, 2012
On a larger scale…
• The blood-brain barrier (BBB) provides a
stable environment for neural function
through a combination of specific ion
channels and transporters which keep the
ionic composition optimal for synaptic
signaling.
• Functions as a protective barrier that shields
the CNS from neurotoxic substances that is
present in the blood.
• Dysfunction of the BBB has been found in
several CNS pathologies such as stroke,
trauma, infectious or inflammatory processes,
MS, HIV, Alzheimer’s, Parkinson’s, epilepsy,
Monday, January 30, 2012
• The BBB is created by the endothelial cells
that form the walls of capillaries.
• There are 3 main barrier sites between the
blood and the brain:
– The BBB proper – created at the level of the
cerebral capillary endothelial cells by tight
junction formation. No brain cell is further
than ~25nm from a capillary so diffusion
distances are short
– The blood-CSF barrier
– The arachnoid barrier – the brain is enveloped
by the arachnoid membrane lying under the
Monday, January 30, 2012
• At all three of the
barriers, the
function is a result
of a physical barrier
formed by tight
junctions, a
transport barrier
formed by specific
transport
mechanisms
mediating solute
flux, and a
metabolic barrier
formed by enzymes
Monday, January 30, 2012
Abbot et al. 200
Tight Junctions
• Tight junctions act
as a fence in the
membrane and
segregate transport
proteins and lipid
rafts, to either the
luminal or abluminal
membrane domain,
and to prevent their
free movement from
one side of the
epithelium to the
Monday, January 30, 2012
Abbot et al.
2009
Selective Transport
• Specific transport
mechanisms must
exist to supply the
brain with just the
right amount of
essential water
soluble nutrients
and metabolites that
are required by
nervous tissue
Monday, January 30, 2012
Abbot et al.
2009
Thank You!!!
References:
• Abbott, N.J., et al., Structure and function of the
blood–brain barrier. Neurobiol. Dis. (2009).
• Armstrong, CM and Hille B. Voltage-gated ion
channels and electrical excitability. Neuron Vol. 20,
pp. 371–380 (1998).
• Cooper EC, Yeh Jan L. Ion Channel Genes and Human
Neurological Disease: Recent Progress, Prospects,
and Challenges. PNAS, Vol. 96, pp.4759-4766 (1999).
• MacKinnon R. Minireview: Potassium Channels. FEBS
Letters 555, pp. 62-65 (2003).
• Mackinnon R., et al. The Structure of the Potassium
Channel: Molecular Basis of K+ Conduction and
Selectivity. Science Vol. 280, pp. 69-77 (1998).
Monday, January 30, 2012