Transcript Voltage-Gated Sodium Channels

Voltage-Gated Sodium Channels
Zhenbo Huang & Brandon Chelette
Membrane Biophysics, Fall 2014
Voltage-gated Sodium Channels
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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
Area under
curve analysis
of postsynaptic
current
Post synaptic
currents
Behavioral Confirmation/Followup/Investigation
• Mice subjected to battery of behavioral tests
that test odor-guided behaviors.
• Consensus: inability to detect odors
Behavioral Confirmation/Followup/Investigation
Innate Olfactory Preference Test
Behavioral Confirmation/Followup/Investigation
Odor avoidance behavior test
Black circle = TMT (fox odor)
Behavioral Confirmation/Followup/Investigation
1. Novel odor investigation
2. Odor learning
3. Odor discrimination
Behavioral Confirmation/Followup/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
William A. Catterall, 2000
http://courses.washington.edu/conj/membrane/nachan.htm
Why activation of sodium channel is quicker than potassium channels?
NavAb
Payandeh et al., 2011
KvAP
D. Peter Tieleman, 2006
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.
Acceleration of VS Movement in Mammalian
Nav Channels by the β1 Subunit
Gating current
Ionic current
Clay M. Armstrong (2008), Scholarpedia,
3(10):3482.
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-Control
Residues in Voltage Sensors
Mechanisms conserve in a evolutionary-distant VS
Ciona Intestinalis
voltage-sensitive
phosphatase(Ci-VSP)
The Sodium Channel Accessory Subunit Navβ1 Regulates
Neuronal Excitability through Modulation of Repolarizing
Voltage-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
William A. Catterall, 2000
Navβ1 is identified in mouse brain Kv4.2 channel complexes
Mass spectrometric analyses
Navβ1 coimmunoprecipitates with Kv4.2
Navβ1 increases Kv4.2-encoded current densities
Coexpression with Navβ1 increases total and cellsurface 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