Voltage-Gated Sodium Channels: Structure, Diversity & Pathologies

1. Voltage-Gated Sodium Channels Zhenbo Huang & Brandon Chelette Membrane Biophysics, Fall 2014

2. Voltage-gated Sodium Channels • Historical importance • Structure • Biophysical importance • Diversity • Associated pathologies

3. 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

4. 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

5. Structure

6. 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

7. Biophysical Importance

8. 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)

9. Biophysical Importance

10. 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

11. Diversity

12. Associated Pathologies

13. Summary • Incredibly important group of membrane channel proteins • Widely expressed throughout many tissues and involved in many functions

14. Loss-of-function mutations in sodium channel Nav1.7 cause anosmia Weiss, et al. 2011. Nature

15. 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?

16. 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)

17. 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)

18. 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

19. Nav1.7 in Olfactory Sensory Neurons Human olfactory epithelium of normal, unaffected adults

20. Creating Nav1.7 KO mice Nav1.7 expression in mouse OSNs

21. Creating Nav1.7 KO mice Nav1.7 expression in mouse olfactory bulb and main olfactory epithelium

22. 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

23. 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)

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

25. 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.

26. 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

27. 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

28. 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)

29. Investigation of Biophysical Role of Nav1.7 Post synaptic potentials Area under curve analysis of postsynaptic current Post synaptic currents

30. Behavioral Confirmation/Follow-up/Investigation • Mice subjected to battery of behavioral tests that test odor-guided behaviors. • Consensus: inability to detect odors

31. Behavioral Confirmation/Follow-up/Investigation Innate Olfactory Preference Test

32. Behavioral Confirmation/Follow-up/Investigation Odor avoidance behavior test Black circle = TMT (fox odor)

33. Behavioral Confirmation/Follow-up/Investigation • Novel odor investigation • Odor learning • Odor discrimination

34. Behavioral Confirmation/Follow-up/Investigation Pup retrieval ability of females (likely depends on olfactory cues)

35. 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

36. 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

37. William A. Catterall, 2000 http://courses.washington.edu/conj/membrane/nachan.htm

38. Why activation of sodium channel is quicker than potassium channels? NavAb KvAP Payandeh et al., 2011 D. Peter Tieleman, 2006

39. 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.

40. 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

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

42. Two Speed-Control Residues in Voltage Sensors

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

45. A Mechanism for the Speed-Control Residues in Voltage Sensors

46. Mechanisms conserve in a evolutionary-distant VS Ciona Intestinalis voltage-sensitive phosphatase(Ci-VSP)

47. The Sodium Channel Accessory Subunit Navβ1 RegulatesNeuronal Excitability through Modulation of RepolarizingVoltage-Gated KChannels 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

48. William A. Catterall, 2000

49. Navβ1 is identified in mouse brain Kv4.2 channel complexes Mass spectrometric analyses

50. Navβ1 coimmunoprecipitates with Kv4.2