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. 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 Na v 1.8; 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 Na v 1.7 cause anosmia Weiss, et al. 2011. Nature

15. Na v 1.7 is necessary for functional nociception SCN9A gene  Na v 1.7 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 Na v 1.7 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. Na v 1.7 in Olfactory Sensory Neurons Loss of olfactory capabilities can only be attributed to loss-of-function mutation in SCN9A if Na v 1.7 is expressed somewhere in the olfactory system. But at what junction? First guess: OSNs

19. Na v 1.7 in Olfactory Sensory Neurons Human olfactory epithelium of normal, unaffected adults

20. Creating Na v 1.7 KO mice Na v 1.7 expression in mouse OSNs

21. Creating Na v 1.7 KO mice Na v 1.7 expression in mouse olfactory bulb and main olfactory epithelium

22. Creating Na v 1.7 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 Na v 1.7 KO mice Okay, so Na v 1.7 is highly expressed in the olfactory sensory neurons. Especially in the olfactory nerve layer and the glomerular layer. Tissue selective KO of Na v 1.7 in OSNs using lox-cre system under control of OMP promoter. Cre recombinase-mediated deletion of Na v 1.7 in OMP-positive cells (which includes all OSNs)

24. Creating Na v 1.7 KO mice Na v 1.7 -/- mice loss of immunoreactivity in OB and MOE

25. Investigation of Biophysical Role of Na v 1.7 Voltage clamp MOE tissue of Na v 1.7 -/- and Na v 1.7 +/- Both resulted in TTX-sensitive currents in response to step depolarizations.

26. Investigation of Biophysical Role of Na v 1.7 OSNs of Na v 1.7 -/- mice show significant sodium current Only a ~20% reduction of current compared to Na v 1.7 +/- OSNs

27. Investigation of Biophysical Role of Na v 1.7 Na v 1.7 -/- OSNs are still capable of generating odor-evoked action potentials “Loose-patch” recording of OSN dendritic knobs

28. Investigation of Biophysical Role of Na v 1.7 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 Na v 1.7 Post synaptic potentials Post synaptic currents Area under curve analysis of postsynaptic current

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 1.Novel odor investigation 2.Odor learning 3.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 Na v 1.7 gene leads to loss of olfactory capabilities in humans and in KO mice. Since OSNs and Mitral cells are still electrically functional, Na v 1.7 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. http://courses.washington.edu/conj/membrane/nachan.htm William A. Catterall, 2000

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

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. 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

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

47. The Sodium Channel Accessory Subunit Navβ1 Regulates Neuronal 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

48. William A. Catterall, 2000

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

50. Navβ1 coimmunoprecipitates with Kv4.2

52. Navβ1 increases Kv4.2-encoded current densities

53. Coexpression with Navβ1 increases total and cell- surface Kv4.2 protein expression

54. Acute knockdown of Navβ1 decreases I A densities in cortical neurons

55. Loss of Navβ1 prolongs action potentials and increases repetitive firing in cortical pyramidal neurons

56. Navβ1 increases the stability of Kv4.2