Selective Permeability
Jackie Bonds
January 23, 2012
Neus 586
Selective Permeability – What is it?
• 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.
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
• Mechano-sensitive channels
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 voltage-gated Na+
channels to open
• Repolarization is the closing
of Na+ channels, increasing
the relative permeability to K+
What properties govern the
specificity of channels? How can this
be studied? How can we understand
this in terms of dysfunction and
disease?
Techniques
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X-ray crystallography
Electron Density Maps
Pharmacology
Genetics
– Sequencing
– Site-directed mutagenesis
• Patch-clamping
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 allow ions to pass when they
occupy particular sites in the membrane.”
Visualization of the channel
• Structure and function has been studied using Xray 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 within
the filter
General Features of a VGIC
SF = Selectivity Filter
VS = Voltage Sensor
OV = Outer Vestibule
IV = Inner Vestibule
Armstrong et al. 1998
Voltage Sensor (S4 Segment)
• Cysteine mutagenesis and cysteine labeling with thiolreactive 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
response to a positive change of membrane potential
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
diffusion limit.
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 surface
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 charged Cterminus toward the ion
pathway
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 overcome the attractive forces
between K+ ions and the selectivity filter
• 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 that is observed in the
central cavity
MacKinnon. 2003
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 permeant ions in the
pore using electron density maps
MacKinnon et al. 1998
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.
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
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 strong interaction
between ion and protein. This further allows for rapid conduction while still
favoring high selectivity.
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)
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
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
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
• Altered Na+ channel function has been linked
to epilepsy
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 exhibit
slow channel openings and closings
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, brain tumors, pain, and
glaucoma
• 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 dura
• 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
metabolizing molecules
in transit).
Abbot et al. 2009
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 other
in order to preserve the
polarity of the barrier.
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
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).