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…”  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  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. Regulation and Modulation in Na Channels  Phosphorylation effects  Mutations in ball- and-chain affect inactivation speed  Cleavage of any part of Na channel protein  Drugs can be used as modulators  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)

7. Pharmacology (i.e. drugs of choice)  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  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 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)  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. Structural Drug Use  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  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

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  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  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. ENaC in kidney, colon, and lungs  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)  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)

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

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  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?  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)  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. What are nonselective cation channels?  Obvious answer…  However, most NCCs are known for fluxing Ca 2+  Mostly due to chemical gradient of Ca outside of cell  Still flux Na +, K +  Obvious answer…  However, most NCCs are known for fluxing Ca 2+  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

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  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. Structure  TRP channels have 6 transmembrane segments (similar to K v 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  TRP channels have 6 transmembrane segments (similar to K v 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

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], PIP 2 -TRPV [Nilius et al., 2007])  Many of these channels are also activated by Ca2+ binding (Amaral and Pozzo-Miller, 2007)

27. 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.  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  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

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  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  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. Paper Summary  BDNF increases density of dendritic spines on hippocampal neurons (CA1)  Works via a TRPC3 conductance  Uses TrkB receptors, phospholipase C, others  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

35. Summation  Na channels have multiple locations, uses, responses  Well-studied  Structure still not elucidated  Isoforms part of historical work  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