Transcript Sodium Channels and Nonselective Cation Channels

Sodium Channels and
Nonselective Cation Channels
An Introduction
Corthell, 2007
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 (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
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-13/1309.jpg
Ligand-Gated
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-13/1323.jpg
Regulation and Modulation in
Na Channels
 Phosphorylation
effects
 Mutations in balland-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)
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
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)
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
 Other drugs used to
identify other
channel proteins as
well as their
receptor sites
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
 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)
Epithelial Na Channel (ENaC)
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)
 Affected by aldosterone
and vasopressin
 Alter rate of insertion,
degradation, recycling of
channels
 Helped identify channel
recycling by clathrinmediated endocytosis
(Shimkets et al., 1997)
Nicotinic Acetylcholine
Receptor (nAchR)
Model of the ligandbinding domain
Mature muscle
expresses
different subunits
than fetal muscle
http://s12ap550.biop.ox.ac.uk:8078/dynamite_html/gallery_files/nAChR_covariance_lines_small.p
ng
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 patchclamp 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…
 …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
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?
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)
Stretch Receptors
www.unm.edu/~toolson/ pacinian_corpuscle.gif
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+
 Not necessarily a
‘universal’ structure
like Na or K
channels
 Depends on
sequence homology,
location of channel
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
Structure
 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 homoand heterotetramers
 Ankyrin repeats (33
a.a.s) crucial for
some subunits to
assemble
TRPC3 structure (proposed)
Mio et al., 2007
 TRP channels are
 Many of these
known to have many
channels are also
different ligands
activated by Ca2+
(capsaicin-TRP
binding (Amaral and
relative VR1 [Cesare
Pozzo-Miller, 2007)
and McNaughton,
1996, 1997], PIP2TRPV [Nilius et al.,
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
 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
 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
 BDNF application alters amount of TRPC3 on
surface
 Spines affected by
different drugs,
including TRPC
inhibitors
 Spines counted
Paper Summary
 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 BDNFmediated dendritic
remodeling
Summation
 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