Voltage-Gated Sodium Channels

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Voltage-Gated Sodium Channels

• Zhenbo Huang Brandon Chelette
• Membrane Biophysics, Fall 2014

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Voltage-gated Sodium Channels

• Historical importance
• Structure
• Biophysical importance
• Diversity
• Associated pathologies

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Historical importance

• Channels that allowed Hodgkin and Huxley to
  perform their seminal work in the 1950s.
• Evolutionarily ancient
• Catalyst for a large shift in research focus
• Led to the discovery and characterization of many
  more ion channel proteins

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Structure

• Consists of an a subunit and one or two
  associated ß subunit(s).
• The a subunit is sufficient to form a functioning
  sodium channel
• ß subunits alter the kinetics and voltage
  dependence of the channel

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Structure
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Biophysical Importance

• Responsible for initiation of action potential
• Open in response to depolarization and activate
  quickly
• Quickly inactivate
• Allows for patterned firing of action potentials
• Firing pattern signal

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Biophysical Importance
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Biophysical Importance

• Not solely voltage-gated
• Can be modulated by a handful of
  neurotransmitters (ACh, 5-HT, DA, others)
• GPCR ? PKA PKC ? phosphorylation of
  intracellular loop ? reduced channel activity
  (except in Nav1.8 activity is enhanced)

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Biophysical Importance
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Diversity

• 10 different a subunit genes
• Spatial expression
• Temporal expression
• Gating kinetics
• 4 different ß subunits
• ß1 and ß3 non-covalently associated
• ?2 and ß4 disulfide bond

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Diversity
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Associated Pathologies
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Summary

• Incredibly important group of membrane channel
  proteins
• Widely expressed throughout many tissues and
  involved in many functions

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Loss-of-function mutations in sodium channel
Nav1.7 cause anosmia
Weiss, et al. 2011. Nature
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Nav1.7 is necessary for functional nociception

• SCN9A gene ? Nav1.7 a-subunit
• Loss-of-function mutation identified in three
  individuals with chronic analgesia
  (channelopathy-associated insensitivity to pain
  CAIP)
• What about other sensory modalities?

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Role of Nav1.7 in Human Olfaction

• Same subjects from earlier nociception studies
• First subject assessed via University of
  Pennsylvania Smell Identification Test
• Pair of siblings and parents assessed with
  sequence of odors (balsamic vinegar, orange,
  mint, perfume, water, and coffee)

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Results of Olfactory Assessment in CAIP subjects
First subject did not identify any odors in UPSIT

• Siblings could not identify any odors presented
• Parents correctly identified each odor in
  seqeunce (as well as reporting no odor when
  presented with water as control)

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Nav1.7 in Olfactory Sensory Neurons

• Loss of olfactory capabilities can only be
  attributed to loss-of-function mutation in SCN9A
  if Nav1.7 is expressed somewhere in the olfactory
  system. But at what junction?
• First guess OSNs

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Nav1.7 in Olfactory Sensory Neurons
Human olfactory epithelium of normal, unaffected
adults
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Creating Nav1.7 KO mice
Nav1.7 expression in mouse OSNs
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Creating Nav1.7 KO mice
Nav1.7 expression in mouse olfactory bulb and
main olfactory epithelium
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Creating Nav1.7 KO mice
High immunoreactivity in the olfactory nerve
layer and glomerular layer of olfactory bulb
Also high immunoreactivity in olfactory sensory
neuron axon bundles of the main olfactory
epithelium
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Creating Nav1.7 KO mice

• Okay, so Nav1.7 is highly expressed in the
  olfactory sensory neurons. Especially in the
  olfactory nerve layer and the glomerular layer.
• Tissue selective KO of Nav1.7 in OSNs using
  lox-cre system under control of OMP promoter.
• Cre recombinase-mediated deletion of Nav1.7 in
  OMP-positive cells (which includes all OSNs)

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Creating Nav1.7 KO mice
Nav1.7 -/- mice loss of immunoreactivity in OB
and MOE
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Investigation of Biophysical Role of Nav1.7

• Voltage clamp MOE tissue of Nav1.7 -/- and Nav1.7
  /-
• Both resulted in TTX-sensitive currents in
  response to step depolarizations.

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Investigation of Biophysical Role of Nav1.7
OSNs of Nav1.7 -/- mice show significant sodium
current
Only a 20 reduction of current compared to
Nav1.7 /- OSNs
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Investigation of Biophysical Role of Nav1.7
Nav1.7 -/- OSNs are still capable of generating
odor-evoked action potentials Loose-patch
recording of OSN dendritic knobs
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Investigation of Biophysical Role of Nav1.7
Nerve stimulation leads to postsynaptic response
in mitral cell in /- but not -/- (patch clamp,
whole cell) Direct current injection from
pipette produced normal APs in both /- and
-/- (current clamp, whole cell)
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Investigation of Biophysical Role of Nav1.7
Post synaptic potentials
Area under curve analysis of postsynaptic current
Post synaptic currents
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Behavioral Confirmation/Follow-up/Investigation

• Mice subjected to battery of behavioral tests
  that test odor-guided behaviors.
• Consensus inability to detect odors

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Behavioral Confirmation/Follow-up/Investigation
Innate Olfactory Preference Test
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Behavioral Confirmation/Follow-up/Investigation
Odor avoidance behavior test
Black circle TMT (fox odor)
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Behavioral Confirmation/Follow-up/Investigation

1. Novel odor investigation
2. Odor learning
3. Odor discrimination

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Behavioral Confirmation/Follow-up/Investigation
Pup retrieval ability of females (likely depends
on olfactory cues)
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Conclusions

• Loss-of-function mutation in Nav1.7 gene leads to
  loss of olfactory capabilities in humans and in
  KO mice.
• Since OSNs and Mitral cells are still
  electrically functional, Nav1.7 must be critical
  for propagation of the signal in the glomerular
  layer

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Molecular Bases for the Asynchronous Activation
of Sodium and Potassium Channels Required for
Nerve Impulse Generation  Jérôme J. Lacroix,
Fabiana V. Campos, Ludivine Frezza, Francisco
Bezanilla  Neuron  Volume 79, Issue 4, Pages
651-657 (August 2013) DOI 10.1016/j.neuron.2013.
05.036
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William A. Catterall, 2000
http//courses.washington.edu/conj/membrane/nachan
.htm
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Why activation of sodium channel is quicker than
potassium channels?
NavAb
KvAP
Payandeh et al., 2011
D. Peter Tieleman, 2006
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What we have know

• Opening Nav channels requires the rearrangement
  of only three VSs, while pore opening in Kv
  channels typically requires the rearrangement of
  four
• It is known that the main factor underlying fast
  activation of Nav channels is the rapid
  rearrangement of their VS.

What is still unknown

• The molecular bases for the kinetic differences
  between voltage sensors of Na and K channels
  remain unexplained.

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Acceleration of VS Movement in Mammalian Nav
Channels by the ß1 Subunit
Gating current
Ionic current
Clay M. Armstrong (2008), Scholarpedia,
3(10)3482.
http//courses.washington.edu/conj/membrane/nachan
.htm
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Acceleration of VS Movement in Mammalian Nav
Channels by the ß1 Subunit
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Two Speed-Control Residues in Voltage Sensors
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Hydrophilic Conversion of Speed-Control Residues
in Nav1.4 DIV Accelerates Fast Inactivation
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A Mechanism for the Speed-Control Residues in
Voltage Sensors
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Mechanisms conserve in a evolutionary-distant VS
Ciona Intestinalis voltage-sensitive
phosphatase(Ci-VSP)
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The Sodium Channel Accessory Subunit Navß1
RegulatesNeuronal Excitability through
Modulation of RepolarizingVoltage-Gated K
Channels
Celine Marionneau, Yarimar Carrasquillo, Aaron J.
Norris, R. Reid Townsend, Lori L. Isom, Andrew J.
Link, and Jeanne M. Nerbonne

• The Journal of Neuroscience, April 25, 2012
  32(17)5716 5727

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William A. Catterall, 2000
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Navß1 is identified in mouse brain Kv4.2 channel
complexes
Mass spectrometric analyses
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Navß1 coimmunoprecipitates with Kv4.2
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Navß1 increases Kv4.2-encoded current densities
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Coexpression with Navß1 increases total and
cell-surface Kv4.2 protein expression
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Acute knockdown of Navß1 decreases IA densities
in cortical neurons
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Loss of Navß1 prolongs action potentials and
increases repetitive firing in cortical pyramidal
neurons
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Navß1 increases the stability of Kv4.2