Voltage-Gated Sodium Channels Zhenbo Huang & Brandon Chelette Membrane Biophysics, Fall 2014.
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Transcript of Voltage-Gated Sodium Channels Zhenbo Huang & Brandon Chelette Membrane 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
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
• 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)
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
Summary
• Incredibly important group of membrane channel proteins
• Widely expressed throughout many tissues and involved in many functions
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
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)
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
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
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
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
Loss of Navβ1 prolongs action potentials and increases repetitive firing in cortical pyramidal neurons