Sodium Channels and Nonselective Cation Channels An Introduction Corthell, 2007
Dec 23, 2015
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
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)
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)
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
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