Sodium Channels and Nonselective Cation

Channels

Sodium Channels and Nonselective Cation

ChannelsAn IntroductionAn Introduction

Corthell, 2007

OutlineOutline 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…”

Sodium (Na) Channel Types

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

Voltage-GatedVoltage-Gated

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

Ligand-GatedLigand-Gated

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

Regulation and Modulation in Na Channels

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)

NO modulates Na currents (Ribeiro et al., 2007) NO donors reduce

peak Na current

ENaC modulated by accessory proteins (Gormley et al., 2003)

Pharmacology (i.e. drugs of choice)

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 onceSaxitoxin

www.chemfinder.com

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)

Structural Drug UseStructural 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

Other drugs used to identify other channel proteins as well as their receptor sites

Na Channel StructureNa 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

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)

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)

Epithelial Na Channel (ENaC)

Epithelial Na Channel (ENaC)

ENaC in kidney, colon, and lungs

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)

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)

Nicotinic Acetylcholine Receptor (nAchR)

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.png

Mature muscle expresses different subunits than fetal muscle

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

al., 2007)

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

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

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

…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)

…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)

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

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

Paper SummaryPaper 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?

Nonselective Cation Channels

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)

Stretch ReceptorsStretch Receptors

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

What are nonselective cation channels?

What are nonselective cation channels?

Obvious answer… However, most

NCCs are known for fluxing Ca2+

Mostly due to chemical gradient of Ca outside of cell

Still flux Na+, K+

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

Not necessarily a ‘universal’ structure like Na or K channels Depends on

sequence homology, location of channel

Transient Receptor Potential (TRP) Channels

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

StructureStructure

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

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

Different subunits: made up of homo- and heterotetramers Ankyrin repeats

(33 a.a.s) crucial for some subunits to assemble

TRPC3 structure (proposed)

Mio et al., 2007

TRP channels are known to have many different ligands (capsaicin-TRP relative VR1 [Cesare and McNaughton, 1996, 1997], PIP2-TRPV [Nilius et al., 2007])

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)

Many of these channels are also activated by Ca2+ binding (Amaral and Pozzo-Miller, 2007)

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.

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

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

Kept in serum-free media to avoid effects of serum nutrients

Slowly activating, sustained current Different than

other Trk receptor cation fluxes

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

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

Current is not blocked by saxitoxin

TRPC currents expressed in hippocampal neurons

Current is not blocked by saxitoxin

TRPC currents expressed in hippocampal neurons

BDNF application alters amount of TRPC3 on surface

BDNF application alters amount of TRPC3 on surface

Spines affected by different drugs, including TRPC inhibitors

Spines affected by different drugs, including TRPC inhibitors

Spines counted Spines counted

Paper SummaryPaper 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

Therefore, TRPC3 channels are mediators of BDNF-mediated dendritic remodeling

SummationSummation

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

Nonselective cation channels are found in most sensory systems Transduction

channels or TRP channels

Many different purposes, depending on host cell