voltage-gated sodium channels arij daou & andrea stathopoulos membrane biophysics fall 2011

Voltage-Gated Sodium Channels

Arij Daou & Andrea StathopoulosMembrane Biophysics

Fall 2011

Na+ channels in excitable membranes

Activation-Inactivation-Deactivation

Location of Na+ channelsName 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 2011Volkers, L, Kahlig, KM, Verbeek, NE, Das, JHG, van Kempen MJA, Stroink, H, et al.

GEFS+ and DSGenetic Epilepsy with Febrile seizures-plus

• Seizures during infancy associated with a rise in body temperature

• May experience afebrile seizures later in life

Dravet syndrome• Also associated with fever-

induced 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 voltage-gated sodium currents and Nav1.7

sodium channels

Neuroscience Letters 2010He, B & Soderlund, DM

HEK293 cells

• Commonly used for heterologous expression– Overexpressing a foreign protein in a simple-to-

use 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.Section of Neurobiology, University of Texas at Austin, Texas2.Institute of Cell and Molecular Biology, University of Texas at Austin, Texas3.Department of Pharmacology and Toxicology, Stark Neurosciences Research Institute, Indiana4.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

• 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 α-helix 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

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 2011Weiss, 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• 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.