Voltage-Gated Sodium Channels

Zhenbo Huang & Brandon CheletteMembrane Biophysics, Fall 2014

Voltage-gated Sodium Channels

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

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

Structure

• Consists of an α subunit and one or two associated β subunit(s).

• The α subunit is sufficient to form a functioning sodium channel

• β subunits alter the kinetics and voltage dependence of the channel

Structure

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

Biophysical Importance

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)

Biophysical Importance

Diversity

• 10 different α subunit genes– Spatial expression– Temporal expression– Gating kinetics

• 4 different β subunits– β1 and β3: non-covalently associated– Β2 and β4: disulfide bond

Diversity

Associated Pathologies

Summary

• Incredibly important group of membrane channel proteins

• Widely expressed throughout many tissues and involved in many functions

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia

Weiss, et al. 2011. Nature

Nav1.7 is necessary for functional nociception

• SCN9A gene Nav1.7 α-subunit

• Loss-of-function mutation identified in three individuals with chronic analgesia (channelopathy-associated insensitivity to pain = CAIP)

• What about other sensory modalities?

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)

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)

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

Nav1.7 in Olfactory Sensory Neurons

Human olfactory epithelium of normal, unaffected adults

Creating Nav1.7 KO mice

Nav1.7 expression in mouse OSNs

Creating Nav1.7 KO mice

Nav1.7 expression in mouse olfactory bulb and main olfactory epithelium

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

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)

Creating Nav1.7 KO mice

Nav1.7 -/- mice loss of immunoreactivity in OB and MOE

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.

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

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

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)

Investigation of Biophysical Role of Nav1.7

Post synaptic potentials

Post synaptic currents

Area under curve analysis of postsynaptic current

Behavioral Confirmation/Follow-up/Investigation• Mice subjected to battery of behavioral tests

that test odor-guided behaviors.• Consensus: inability to detect odors

Behavioral Confirmation/Follow-up/Investigation

Innate Olfactory Preference Test

Behavioral Confirmation/Follow-up/Investigation

Odor avoidance behavior test

Black circle = TMT (fox odor)

Behavioral Confirmation/Follow-up/Investigation

1. Novel odor investigation2. Odor learning3. Odor discrimination

Behavioral Confirmation/Follow-up/Investigation

Pup retrieval ability of females

(likely depends on olfactory cues)

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

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

http://courses.washington.edu/conj/membrane/nachan.htm

William A. Catterall, 2000

Payandeh et al., 2011

NavAb

D. Peter Tieleman, 2006

KvAP

Why activation of sodium channel is quicker than potassium channels?

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.

Clay M. Armstrong (2008), Scholarpedia, 3(10):3482.

Acceleration of VS Movement in Mammalian Nav Channels by the β1 Subunit

Gating current

Ionic current

http://courses.washington.edu/conj/membrane/nachan.htm

Acceleration of VS Movement in Mammalian Nav Channels by the β1 Subunit

Two Speed-Control Residues in Voltage Sensors

Hydrophilic Conversion of Speed-Control Residues in Nav1.4 DIV Accelerates Fast Inactivation

A Mechanism for the Speed-ControlResidues in Voltage Sensors

Ciona Intestinalis voltage-sensitive phosphatase(Ci-VSP)

Mechanisms conserve in a evolutionary-distant VS

The Sodium Channel Accessory Subunit Navβ1 RegulatesNeuronal Excitability through Modulation of Repolarizing

Voltage-Gated K Channels

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

Celine Marionneau, Yarimar Carrasquillo, Aaron J. Norris, R. Reid Townsend, Lori L. Isom, Andrew J. Link, and Jeanne M. Nerbonne

William A. Catterall, 2000

Mass spectrometric analyses

Navβ1 is identified in mouse brain Kv4.2 channel complexes

Navβ1 coimmunoprecipitates with Kv4.2

Navβ1 increases Kv4.2-encoded current densities

Coexpression with Navβ1 increases total and cell-surface Kv4.2 protein expression

Acute knockdown of Navβ1 decreases IA densities in cortical neurons

Loss of Navβ1 prolongs action potentials and increases repetitive firing in cortical pyramidal neurons

Navβ1 increases the stability of Kv4.2