Sodium Channels and Nonselective Cation Channels An Introduction

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Sodium Channels and Nonselective Cation Channels An Introduction Corthell, 2007

Sodium Channels and Nonselective Cation Channels An Introduction Corthell, 2007

Outline w Sodium Channels w Types w Regulatory mechanisms (a few) w Pharmacology (and

Outline w Sodium Channels w Types w Regulatory mechanisms (a few) w Pharmacology (and what it shows us) w Structure w Paper-”Role of hydrophobic residues…” w Nonselective Cation Channels w Where they are w What they are w TRP channels-well-characterized w Paper-”TRPC 3 Channels Are Necessary…”

Sodium (Na) Channel Types w Voltage-gated Na Channels w Include ‘voltage sensor’ on protein

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

Voltage-Gated http: //www. mun. ca/biology/desmid/brian/BIOL 2060 -13/1309. jpg

Voltage-Gated http: //www. mun. ca/biology/desmid/brian/BIOL 2060 -13/1309. jpg

Ligand-Gated http: //www. mun. ca/biology/desmid/brian/BIOL 2060 -13/1323. jpg

Ligand-Gated http: //www. mun. ca/biology/desmid/brian/BIOL 2060 -13/1323. jpg

Regulation and Modulation in Na Channels w Phosphorylation w NO modulates Na effects currents

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

Pharmacology (i. e. drugs of choice) w Saxitoxin (STX), from red tide, used to

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

w Drugs bind to receptors w Can be used to count receptors, block channels

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

Structural Drug Use w TTX and STX used to w Other drugs used to

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

Na Channel Structure w 6 transmembrane domains (S 1 -S 6) w 4 repeats

Na Channel Structure w 6 transmembrane domains (S 1 -S 6) w 4 repeats (Domain 1 -4) w Has , , and subunits w subunit responsible for pore w P-loop as selectivity filter

w Single linked protein makes up ion channel w P-loop reflects speed of inactivation

w Single linked protein makes up ion channel w P-loop reflects speed of inactivation w , subunits modify channel function but are not essential to create the pore w Ligand-gated channels do not have voltage sensor, but ligand binding site w Voltage gated channels have voltage sensor on S 4 in each domain w Speculation: domain sensors have special functions (Kuhn and Greef, 1999)

Epithelial Na Channel (ENa. C)

Epithelial Na Channel (ENa. C)

ENa. C in kidney, colon, and lungs w Kidney: ENa. C aids in Na.

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

Nicotinic Acetylcholine Receptor (n. Ach. R) Model of the ligandbinding domain Mature muscle expresses

Nicotinic Acetylcholine Receptor (n. Ach. R) Model of the ligandbinding domain Mature muscle expresses different subunits than fetal muscle http: //s 12 ap 550. biop. ox. ac. uk: 8078/dynamite_html/gallery_files/n. ACh. R_covariance_lines_small. p ng

Paper: “Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel”

Paper: “Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel” Bendahhou et al. , 2007) w S 4 of each domain is considered the voltage sensor w Major players include Arg and Lys residues occurring every 3 a. a. s and separated by 2 neutral residues w Mutate nonpolar Phe and Leu to Ala w Eliminate steric hindrance w Follow up with patchclamp recording Alter D 1 -D 3, as D 4 S 4 has been studied extensively

w D 1 and D 2 voltage sensor mutations did not result in significantly

w D 1 and D 2 voltage sensor mutations did not result in significantly altered activation/inactivation kinetics…

w …but did alter the activation curve. L 224 A is shifted to a

w …but did alter the activation curve. L 224 A is shifted to a hyperpolarized voltage, enhancing the open state, while L 227 A is shifted to a depolarized voltage (favors closed)

w D 3 mutations led to altered fast inactivation and a voltage shift in

w D 3 mutations led to altered fast inactivation and a voltage shift in inactivation to hyperpolarization

Paper Summary w Hydrophobic residues are also important to the voltage sensor w Need

Paper Summary w Hydrophobic residues are also important to the voltage sensor w Need correct shape w Altering the voltage sensor on D 1 and D 2 alters inactivation/activation kinetics w Mutations on D 3 S 4 alter kinetics and voltage dependence w Leads to idea: perhaps each S 4 responsible for different aspects of channel gating? Do they function independently?

Nonselective Cation Channels w Where? w Across most sensory systems as transduction channels w

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

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

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

What are nonselective cation channels? w Obvious answer… w Not necessarily a ‘universal’ structure

What are nonselective cation channels? w Obvious answer… w Not necessarily a ‘universal’ structure w However, most NCCs like Na or K channels are known for w Depends on fluxing Ca 2+ w Mostly due to chemical gradient of Ca outside of cell w Still flux Na+, K+ sequence homology, location of channel

Transient Receptor Potential (TRP) Channels w Very large gene family-many divisions w TRPM, TRPC,

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

Structure w TRP channels have 6 transmembrane segments (similar to Kv channels) w Between

Structure w TRP channels have 6 transmembrane segments (similar to Kv channels) w Between S 5 and S 6 is believed to be pore w TRP domain: highly conserved 25 a. a. s C-terminal to S 6 w Include 6 invariant a. a. s , called TRP box w Different subunits: made up of homoand heterotetramers w Ankyrin repeats (33 a. a. s) crucial for some subunits to assemble

TRPC 3 structure (proposed) Mio et al. , 2007

TRPC 3 structure (proposed) Mio et al. , 2007

w TRP channels are w Many of these known to have many channels are

w TRP channels are w Many of these known to have many channels are also different ligands activated by Ca 2+ (capsaicin-TRP binding (Amaral and relative VR 1 [Cesare Pozzo-Miller, 2007) and Mc. Naughton, 1996, 1997], PIP 2 TRPV [Nilius et al. , 2007])

Paper-”TRPC 3 Channels Are Necessary for Brain-Derived Neurotrophic Factor to Activate a Nonselective Cationic

Paper-”TRPC 3 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. w BDNF elicits a current that is not blocked by tetrodotoxin or saxitoxin but is blocked by interfering RNA-mediated knockdown of TRPC 3 w BDNF application also increases surface TRPC 3 in cultured hippocampal neurons

w Long-term BDNF exposure leads to various effects on hippocampal neurons w Can modulate

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

w In voltage clamp. K-252 a is a tyrosine kinase inhibitor, showing that the

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

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

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

w BDNF application alters amount of TRPC 3 on surface

w BDNF application alters amount of TRPC 3 on surface

w Spines affected by different drugs, including TRPC inhibitors w Spines counted

w Spines affected by different drugs, including TRPC inhibitors w Spines counted

Paper Summary w BDNF increases density of dendritic spines on hippocampal neurons (CA 1)

Paper Summary w BDNF increases density of dendritic spines on hippocampal neurons (CA 1) w Works via a TRPC 3 conductance w Uses Trk. B receptors, phospholipase C, others w Therefore, TRPC 3 channels are mediators of BDNFmediated dendritic remodeling

Summation w Na channels have multiple locations, uses, responses w Well-studied w Structure still

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