Sodium Channels and Nonselective Cation Channels

1. Sodium Channels and Nonselective Cation Channels An Introduction Corthell, 2007

2. Outline • Sodium Channels • Types • Regulatory mechanisms (a few) • Pharmacology (and what it shows us) • Structure • Paper-”Role of hydrophobic residues…” • Nonselective Cation Channels • Where they are • What they are • TRP channels-well-characterized • Paper-”TRPC3 Channels Are Necessary…”

3. Sodium (Na) Channel Types • Voltage-gated Na Channels • Include ‘voltage sensor’ on protein • Crucial to establish an action potential (AP) • Found in various systems with variant effects and ‘operating voltages’ • Ligand-gated Na Channels • Bind to specific ligand and generate electrical response

4. Voltage-Gated http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-13/1309.jpg

5. Ligand-Gated http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-13/1323.jpg

6. Phosphorylation effects Mutations in ball-and-chain affect inactivation speed Cleavage of any part of Na channel protein Drugs can be used as modulators NO modulates Na currents (Ribeiro et al., 2007) NO donors reduce peak Na current ENaC modulated by accessory proteins (Gormley et al., 2003) Regulation and Modulation in Na Channels

7. Saxitoxin (STX), from red tide, used to count Na channels (Ritchie et al. 1976) Tetrodotoxin (TTX), from fugu puffer fish, local anesthetics also block Na channel flux Local anesthetic: # channels open at once Pharmacology (i.e. drugs of choice) Saxitoxin www.chemfinder.com

8. Drugs bind to receptors • Can be used to count receptors, block channels (ex: identify which current is responsible for some spiking) • Na channel is not perfectly selective • Also permeable to K+ ions, though much less than Na+ (Chandler and Meves, 1965) • Therefore, drug application may not necessarily block one ion completely • Drug responses are variable • Cardiac cells respond less to TTX than skeletal muscle cells (Ritchie and Rogart, 1977; Cohen et al., 1981)

9. TTX and STX used to identify Na channel proteins (Henderson and Wang, 1972) Irradiated TTX and STX used as markers for bound portions of protein Other drugs used to identify other channel proteins as well as their receptor sites Structural Drug Use

10. Na Channel Structure • 6 transmembrane domains (S1-S6) • 4 repeats (Domain 1-4) • Has , , and  subunits •  subunit responsible for pore • P-loop as selectivity filter

11. Single linked protein makes up ion channel P-loop reflects speed of inactivation ,  subunits modify channel function but are not essential to create the pore Ligand-gated channels do not have voltage sensor, but ligand binding site Voltage gated channels have voltage sensor on S4 in each domain Speculation: domain sensors have special functions (Kuhn and Greef, 1999)

12. Epithelial Na Channel (ENaC)

13. Kidney: ENaC aids in NaCl reabsorption Maintains body NaCl balance and blood pressure (Garty and Benos, 1988) Lungs: aids in fluid clearance from alveolar space Maintains normal gas exchange in lungs (Matalon and O’Brodovich, 1999) Affected by aldosterone and vasopressin Alter rate of insertion, degradation, recycling of channels Helped identify channel recycling by clathrin-mediated endocytosis (Shimkets et al., 1997) ENaC in kidney, colon, and lungs

14. Nicotinic Acetylcholine Receptor (nAchR) Model of the ligand-binding domain Mature muscle expresses different subunits than fetal muscle http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/gallery_files/nAChR_covariance_lines_small.png

15. Paper: “Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel” Bendahhou et al., 2007) • S4 of each domain is considered the voltage sensor • Major players include Arg and Lys residues occurring every 3 a.a.s and separated by 2 neutral residues • Mutate nonpolar Phe and Leu to Ala • Eliminate steric hindrance • Follow up with patch-clamp recording Alter D1-D3, as D4 S4 has been studied extensively

16. D1 and D2 voltage sensor mutations did not result in significantly altered activation/inactivation kinetics…

17. …but did alter the activation curve. L224A is shifted to a hyperpolarized voltage, enhancing the open state, while L227A is shifted to a depolarized voltage (favors closed)

18. D3 mutations led to altered fast inactivation and a voltage shift in inactivation to hyperpolarization

19. Paper Summary • Hydrophobic residues are also important to the voltage sensor • Need correct shape • Altering the voltage sensor on D1 and D2 alters inactivation/activation kinetics • Mutations on D3 S4 alter kinetics and voltage dependence • Leads to idea: perhaps each S4 responsible for different aspects of channel gating? Do they function independently?

20. Nonselective Cation Channels • Where? • Across most sensory systems as transduction channels • Examples: retinal rods, hair cells, Pacinian corpuscle, spindle organs, taste cells (amino acid taste), nociception • TRP channels extensively studied • Broad family of nonselective cation channels • In brain, aiding in spontaneous firing (Kim et al., 2007)

21. Stretch Receptors www.unm.edu/~toolson/ pacinian_corpuscle.gif

22. Obvious answer… However, most NCCs are known for fluxing Ca2+ Mostly due to chemical gradient of Ca outside of cell Still flux Na+, K+ Not necessarily a ‘universal’ structure like Na or K channels Depends on sequence homology, location of channel What are nonselective cation channels?

23. Transient Receptor Potential (TRP) Channels • Very large gene family-many divisions • TRPM, TRPC, TRPV… • Widely expressed in brain (including hippocampus) • Structural similarity, but still many differences between channel structures and functions

24. TRP channels have 6 transmembrane segments (similar to Kv channels) Between S5 and S6 is believed to be pore TRP domain: highly conserved 25 a.a.s C-terminal to S6 Include 6 invariant a.a.s , called TRP box Different subunits: made up of homo- and heterotetramers Ankyrin repeats (33 a.a.s) crucial for some subunits to assemble Structure

25. TRPC3 structure (proposed) Mio et al., 2007

26. TRP channels are known to have many different ligands (capsaicin-TRP relative VR1 [Cesare and McNaughton, 1996, 1997], PIP2-TRPV [Nilius et al., 2007]) Many of these channels are also activated by Ca2+ binding (Amaral and Pozzo-Miller, 2007)

27. BDNF elicits a current that is not blocked by tetrodotoxin or saxitoxin but is blocked by interfering RNA-mediated knockdown of TRPC3 BDNF application also increases surface TRPC3 in cultured hippocampal neurons Paper-”TRPC3 Channels Are Necessary for Brain-Derived Neurotrophic Factor to Activate a Nonselective Cationic Current and to Induce Dendritic Spine Formation” Amaral and Pozzo-Miller, 2007.

28. Long-term BDNF exposure leads to various effects on hippocampal neurons Can modulate synaptic transmission Can change structure of dendrites, spines, and presynaptic terminals Kept in serum-free media to avoid effects of serum nutrients Slowly activating, sustained current Different than other Trk receptor cation fluxes

29. In voltage clamp. K-252a is a tyrosine kinase inhibitor, showing that the BDNF response requires one

30. Current is not blocked by saxitoxin TRPC currents expressed in hippocampal neurons

31. BDNF application alters amount of TRPC3 on surface

32. Spines affected by different drugs, including TRPC inhibitors Spines counted

34. BDNF increases density of dendritic spines on hippocampal neurons (CA1) Works via a TRPC3 conductance Uses TrkB receptors, phospholipase C, others Therefore, TRPC3 channels are mediators of BDNF-mediated dendritic remodeling Paper Summary

35. Na channels have multiple locations, uses, responses Well-studied Structure still not elucidated Isoforms part of historical work Nonselective cation channels are found in most sensory systems Transduction channels or TRP channels Many different purposes, depending on host cell Summation