Transcript Voltage-Gated Sodium Channels

Voltage-Gated Sodium Channels
Arij Daou & Andrea Stathopoulos
Membrane Biophysics
Fall 2011
Na+ channels in excitable membranes
Activation-Inactivation-Deactivation
Location of Na+ channels
Name
Location
Nav1.1
CNS/PNS
Nav1.2
CNS
Nav1.3
CNS
Nav1.4
Skeletal Muscle
Nav1.5
Heart/Denervated skeletal muscle
Nav1.6
CNS/PNS
Nav1.7
PNS
Nav1.8
PNS
Nav1.9
PNS
TRP
Widely expressed
ENaC
Kidneys/Neurons/Lungs
NAChR
CNS/Muscle
Key features of all Nav+ channels
1- Voltage dependent activation.
2- Rapid Inactivation.
3- Selective Ion Conductance
Nav1.1 dysfunction in genetic
epilepsy with febrile seizures-plus or
Dravet syndrome
European Journal of Neuroscience 2011
Volkers, L, Kahlig, KM, Verbeek, NE, Das,
JHG, van Kempen MJA, Stroink, H, et al.
GEFS+ and DS
Genetic Epilepsy with Febrile seizuresplus
• Seizures during infancy
associated with a rise in
body temperature
• May experience afebrile
seizures later in life
Dravet syndrome
• Also associated with feverinduced seizures in early
childhood
• Full “grand mal” seizures
and involuntary twitching
are characteristics
• Slowed development
Role of Na channels in GEFS+ & DS
• Mutations in the  Nav1.1 and β subunits have
been linked to these disorders
• Both gain-of-function and loss-of-function
mutations have been uncovered
Methods
• Blood samples from 7 patients were analyzed
– PCR was used to amplify the DNA, specifically the
coding region for the Nav1.1 channel
• Mutant plasmids were generated to match the
mutations observed in patients
– These  subunits were then expressed in cultured
cells with normal β subunits for
electrophysiological examination
Mutations
http://analytical.wikia.com/wiki/Arginine, http://analytical.wikia.com/wiki/Histidine,
http://smabiology.blogspot.com/2008/10/chapter-four-isomers-and-functional.html
Mutations
• R946C, R946H = nonfunctional channel
– Mutation in the pore loop
• Responsible for ion selectivity
• R859H, R865G = functional channels
– Mutation in S4
• Responsible for voltage-sensing
Electrophysiology Results
• Both mutants open at lower voltages
Electrophysiology Results
• R859H is slower to inactivate, and both
mutants are slower to return from the inactive
to the closed (ready to re-open) state
Electrophysiology Results
• R859H is slower to open • R859H is slow during both
phases of inactivation,
R865G is slow only during
the slow-inactivation phase
Electrophysiology Results
• Both mutants show persistent current
– May be due to incomplete inactivation
– Facilitates repetitive firing
Summary of Results
• Mutant channels:
– Open at lower voltages
– Open slowly
– Inactivate slowly
– Return to closed state more slowly
– Show persistent current
Conclusions
• Dysfunction of sodium channels, even a tiny
change in a single amino acid, can have drastic
effects on not just the functioning of the
individual channel or cell, but on the whole
organism and system of which it is a part
Human embryonic kidney (HEK293)
cells express endogenous voltagegated sodium currents and Nav1.7
sodium channels
Neuroscience Letters 2010
He, B & Soderlund, DM
HEK293 cells
• Commonly used for heterologous expression
– Overexpressing a foreign protein in a simple-touse cell system to better characterize that protein
– Great for understanding individual ion channels,
but it is important to be able to tell your channel
and currents apart from those native to your
expression platform
– Sodium channels have not been characterized in
HEK293 cells, as the sodium-resembling current
has been attributed to another channel type
http://en.wikipedia.org/wiki/Heterologous
Detection of cation currents
Toxin Sensitivity Tests
• It had previously been reported that the
currents observed in HEK293 cells was
sensitive to cadmium block, but not TTX
Toxin Sensitivity Tests
• However, these authors found currents that
were blocked by TTX
– Because the effects of TTX and Cd+2 were additive,
the currents are likely from different channels
Toxin Sensitivity Tests
• TTX also altered the voltage-dependence of
the current, again suggesting that the currents
are from different channels
Toxin Sensitivity Tests
• Tefluthrin slows inactivation and, like TTX,
shifts the voltage-dependence of Na channels
Identification of the Channel
• So far, results suggest that a Na channel is
present in the HEK293 cells
– PCR was used to detect alpha subunits of human
voltage-gated Na channels in the cells
Conclusions
• Nothing was transfected in this study
• TTX and Tefluthrin sensitivity identified a
subpopulation of cation currents as Na currents
from an endogenous voltage-gated Na channel
• Due to the high level of detection for Nav1.7, it is
likely that this isoform of Na channel is present in
HEK293 cells
– This MUST be kept in mind when using heterologous
expression systems
Isolation and Characterization of
CvIV4: A Pain Inducing α-Scorpion
Toxin
Ashlee H. Rowe1, Yucheng Xiao3, Joseph Scales1, Klaus D. Linse2, Matthew P.
Rowe4, Theodore R. Cummins3, Harold H. Zakon1
1.
2.
3.
4.
Section of Neurobiology, University of Texas at Austin, Texas
Institute of Cell and Molecular Biology, University of Texas at Austin, Texas
Department of Pharmacology and Toxicology, Stark Neurosciences Research Institute, Indiana
Department of Biological Sciences, Sam Houston State University, Texas
α-Scorpion Toxins
• Among all species of scorpion, those in the family
Buthidae produce the world’s deadliest venoms.
• The Buthid venom is a mixture of several peptides that
bind different families of ion channels.
• In particular, the α-scorpion toxin binds the Na+ channel,
alters the gating mechanism, inhibits fast inactivation,
and thus prolonging the flow of Na+ ions through the
pore.
α-Scorpion Toxins – Effects and Pain
• The synergistic effect of these toxins is hyper-excitability
of nerve and muscle cells that can cause wide range off
physiological malfunction.
• Even when buthid stings are not fatal, humans report
excruciating pain that may last from several hours to
days.
- Animals sense pain when peripheral nervous system
(nociceptors) are activated and transmit information
about noxious stimuli to the central nervous system.
- The cell bodies of nociceptors are housed in dorsal root
ganglia (DRG), located just outside the spinal cord.
α-Scorpion Toxins – Quantifying Pain
• It had been reported that scorpion venoms
induce paw licking in rodents.
• In this study
– They measured the duration of paw licking by Mus
musculus in response to injections of venom or
venom fractions into their hind paws.
– Determine whether Na+ channel toxins are involved
in generating intense pain produced by buthids.
• Thus, venoms that produce paw licking are
referred to as “painful” or “pain inducing”.
Quantifying the effects of venoms
High Performance Liquid Chromatography
(HPLC) profile of C.vittatus venom fractions
Effect of C.vittatus venom and venom fractions on
paw-licking behavior in Mus musclus
Effect of C.vittatus venom P4 subfractions
CvIV4
Effects of toxin CvIV4 on voltage-gated
sodium channels
• CvIV4 induces pain in mammals.
• CvIV4 is a polypeptide composed of 58 to 76 amino acids
in length (6500-8500 amu) and they contain eight
cysteines that form four disulfide bonds.
• The structural scaffold of this peptide consists of one αhelix and two or three strands of β-sheets, typically
arranged in the order βαββ.
• Pain sensation is regulated, in part, by three VGSC
subtypes (Nav 1.7, Nav 1.8, Nav 1.9) that are expressed
in nociceptors.
• They tested CvIV4 on hNav 1.7 expressed in HEK cells and
on dissociated rat DRG, which expresses all three subtypes.
Effects of toxin CvIV4 on voltage-gated sodium
channels isoforms
Effects of toxin CvIV4 on isoform
NaV1.7
Effects of toxin CvIV4 on isoform
NaV1.7
Effects of toxin CvIV4 on activation and inactivation of
isoform NaV1.2, NaV1.3, NaV1.4 and NaV1.5
Loss-of-function mutations in sodium
channel Nav1.7 cause anosmia
Nature 2011
Weiss, J, Pyrski, M, Jacobi, E, Bufe, B,
Willnecker, V, Schick, B, et al.
Behavioral Assays
Olfactory Bulb Anatomy
http://wires.wiley.com/WileyCDA/WiresArticle/wisId-WSBM85.html
Olfactory Sensory Neurons –
KO characterization
Mitral cell response
Mitral cell response
Summary
• In the absence of Nav 1.7, the OSNs are
electrically active and generate odour-evoked
action potentials but fail to initiate synaptic
signaling to the projection neurons in the
olfactory bulb.
Final Na+ Points
• Na+ channels are critical for the production of
action potentials and normal neural functioning
– This activity is a direct result of their molecular
structure
• Na+ channels are responsible for pain production
in the DRG
– These channels are also central to other organ systems
• Toxins and cellular expression systems are useful
for identifying and characterizing Na+ channels
references
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Catterall, WA (2000) From ionic currents to molecular mechanisms: the structure and function of
voltage-gates sodium channels. Neuron. 26:13-25.
Goldin, AL (2001) Resurgence of sodium channel research. Annu. Rev. Physiol. 63: 871-894.
Clapham, DE, Runnels, LW, & Strubing C (2001) The TRP ion channel family. Nature Rev. Neurosci.
2:387-396.
Mano, I & Driscoll, M (1999). DEG/ENaC channels: a touchy superfamily that watches its salt.
BioEssays. 21(7):568-578.
Volkers, L, Kahlig, KM, Verbeek, NE, Das, JHG, van Kempen, MJA, Stroink, H, et al. (2011) Nav1.1
dysfunction in genetic epilepsy with febrile seizures-plus or Dravet syndrome. Eur. J. Neurosci.
doi: 10.1111/j.1460-9568.2011.07826.x
He, B & Soderlund, DM (2010) Human embryonic kidney (HEK293) cells express endogenous
voltage-gated sodium currents and Nav1.7 sodium channels. Neuroscience Letters. 469:268-272.
Rowe, AH, Xiao, Y, Scales, J, Linse, KD, Rowe, MP, Cummins, TR, et al. (2011) Isolation and
characterization of CvIV4: a pain inducing a scorpion toxin. PLoS ONE. 6(8):e23520.
Weiss, J, Pyrski, M, Jacobi, E, Bufe, D, Willnecker, V, Schick, B, et al. (2011) Loss-of-function
mutations in sodium channel Nav1.7 cause aosmia. Nature. 472:186-192.