Transcript Slide 1

Sodium Channel Structure,
Function, Gating and Involvement
in Disease
David R. Marks, M.Sc.
An Overview
• Sodium Channel Structure
- Current theory and Types of Na+ Channels
• Sodium Channel Function
- Current theory of inactivation
- Modulation
- Pharmacology
- Activation
An Overview Cont’d
• Article 2 – Na+ Channel Gating
• Article 1 - Na+ Channels and
Neurodegenerative Disease
• Article 3 – Na+ channel mutation and
physiology
Sodium Channels - Structure
• Composed of α, β-1 and β-2 subunits, but the large αsubunits carries most of the functional properties
• 4 repeated motifs, each with 6 transmembrane domains
• All linked together
• Contain a voltage “sensor”/ligand binding domain
(method of activation)
• The hydrophobic S4 segment (voltage “sensor”) is found
in all voltage gated Na+ channels and is absent in ligand
gated Na+ channels
• Selectivity filter (shell of hydration)
• Inactivation gate
Cartoon representation of the “typical” voltage-activated sodium channel
Types Of Na+ Channels
• Voltage gated – Changes in membrane
polarity open the channel
• Ligand gated (nicotinic acetylcholine
receptor) – Ligand binding alters
channel/receptor conformation and opens
the pore
• Mechanically gated (stretch receptor) –
Physical torsion or deformation opens the
channel pore
Sodium Channels - Function
• Play a central role in the transmission of action
potentials along a nerve
• Can be in different functional states (3)
-A resting state when it can respond to a
depolarizing voltage changes
-Activated, when it allows flow of Na+ ions
through the
-Inactivated, when subjected to a
“suprathreshold” potential, the channel will not
open
The theory is
that the inactivation gate
“swings” shut, turning off
the channel
Please Keep In Mind
• The structure of the Na+ channel is not
100% solved, hence a “working model” is
drawn based on biophysical,
pharmacological, physiological and
molecular assays
• Zhao (2004) writes “The mechanism of
opening and closing is unknown, but
structural studies suggest…”
Na+ Channel Modulation
• Phosphorylation
• sodium channel function is modulated by
serine/threonine and tyrosine kinases as well as tyrosine
phosphatases (Yu et al, Science 1997)
• Mutation – altered amino acid sequence/structure can
change the biophysical properties of the Na+ channel
• Pharmacology – block Na+ channel to reduce the
conductance
• Proteolysis- (cleavage) Proteases may cleave specific
residues or sequences that inactivate a channel, or
significantly alter the biophysical properties
Why Na+ Channels/Modulation Are
Important
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Neuronal depolarization, Action Potential
Neuronal Excitability
Cardiac Excitability
Muscle Excitability
The basis of neuronal/cardiac/muscular function
relies on the propagation of action potentials,
down axons, sarcolemma, myocardium, as well
as requiring synaptic transmission.
• Differential excitability alters the electrical
conduction/transmission properties of the
“circuit”
Na + Channel
Blockers/Pharmacological Agents
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Tetrodotoxin (TTX)
Amioderone
Lidocaine
Procainamide
Mexilitine
Ketamine
Many, many others
Some Na+ Channels Outside The
Nervous System
• Naf – “Funny Current” in pacemaker cells
of the heart (SA node/ectopic pacemakers)
• Nav in the myocardium, sarcolemma, and
T-tubules and motor endplate
Na+ Channel Activation
• Change in transmembrane potential results in a
conformation change in the Na+ channel
• The four S4 segment alpha helices translocate, thus
leading to the opening of the channel pore
• The energy of the conformational change in the channel
during activation is mediated by the reduction in overall
entropy of the system.
• The voltage sensor is a highly charged sequence of
amino acids that “aligns” itself according to the electrical
field present
• A change in transmembrane potential creates
unfavorable electrodynamic interaction for the voltage
sensor, hence a conformational shift lowers the energy
of the system and creates more favorable conditions
Patch Clamping/Transfection
Transfection
1. Kv1.3 cDNA in Plasmid
2. Lipofectamine complexing
3. Add to Dishes
4. Patch 28-48 hrs after
Transition: A General Overview of
Articles Before Discussion
• From Basic structure/function relationships
to a gating mechanism
• The gating of a bacterial Na+ channel and
application of Na+ channel activation and
biophysical properties
• Article 1 – A gating hinge in Na+ channels:
a molecular switch for electrical signaling
Conserved glycine
In the S6 domain
Proposed conformational shift of A-helix caused by substitution of Proline for G219
Prolines in alpha helices after the first turn (4th residue) cause a kink in the helix.
This kink is caused by proline being unable to complete the
H-bonding chain of the helix and steric or rotamer effects that keep proline from
adapting the prefered helical geometry
Na+ Channel Gating
• Current theory holds that a change in
transmembrane potential “flips” the conformation
of the voltage sensor, thereby opening the
channel pore
• A mutation, G219P, glycine 219 changed to
proline alters the conformation of the S6 domain
• The mutant channel now favors a state much
like the “open” state of a wild-type channel
• NOTE: these bacterial Na+ channels are
homotetramers of identical subunits
Mutation alters the biophysical properties of the channel
The G219P mutant activates significantly earlier (activates at much more
negative voltages) than the wild-type
V ½ : Voltage at which ½ of channels present are in the open state
Comparable to Km in that it is a measure of the ability of a channel to activate
Other mutations to the Na+ channel
Do not exert as significant effects in the
activation (V ½)
Influence of hybrid Na+ channel subunits on gating and biophysical properties
Article 2 - Na+ Channels And
Neurodegenerative Disease
• Overview – Multiple Sclerosis (MS)
displays a remission-relapse course.
Some axons are able to maintain minimal
conduction velocity, while others
degenerate completely.
• Definition: Experimental autoimmune
encephalomytis (EAE) – animal model of
MS
MS can display remission-relapsing
course. This is believed to be the
result of the expression of two
distinct isoforms
of voltage-gated Na+ channels
NaV 1.2/1.6 are expressed over
long distances (> 10μm)
B-amyloid are pepties associated with neurodegenerative diseases, and can accumulate
in fibrillar aggregates
What is Important About This Article
• Nav 1.6 is colocalized with a Na/Ca
exchanger
• Nav 1.2 is NOT colocalized with B-amyloid
proteins
• Nav 1.2 help restore conduction in
demyelinated axons
• Nav 1.6 is seen in degenerating axons
An increase in
NaV1.6 yields an
Increase in Na/Ca
exchangers, elevating
intracellular Ca2+
to harmful levels
Article 3 - Na+ Channels and the
Conduction System of the Heart
• Long QT syndrome – disease where the entire
cycle of excitation-contraction coupling of the
myocardium is prolonged
• Patient had G-A substitution at codon 1763 of
the Nav 1.5 channel gene, which changed a
valine (GTG) to a methionine (ATG)
• This mutation produced a persistently active and
fast recovering Na+ channel
• Mutant was INSENSITIVE to lidocaine
Article 3
• Authors generated a similar mutant by sitedirected mutagenesis
• Examined the mutant in a heterologous
expression system to obtain biophysical and
other properties
The Nav 1.5 V1763M mutant is
Sensitive to TTX, but resistant to
lidocaine
TTX eliminates lidocaine-insensitive current
Why this is important:
Other than traumatic cardiac arrest,
arrhythmias degenerate into ventricular
fibrillation or ventricular tachycardias.
“circus movement” whereby tissue
becomes “hyper-excitable”
Extension and Application of Na+
Channel Properties and Function
Relating to Article 3
Advanced Cardiac Life Support
(ACLS) Targets Na+ Channels Extensively
• “Please Shock Shock Shock, Everybody
Shock, And Lets Make Patients Better”
The purpose of defibrillation of ventricular arrhythmias is to apply a controlled electrical
shock to the heart, which leads to depolarization of the entire electrical conduction
system of the heart. When the heart repolarizes, the normal electrical conduction
may restore itself
Depolarization theoretically inactivates all voltage-gated Na+ channels, and allows
Voltage-gated potassium channels to activate, and help hyperpolarize the membrane
+40 mv
-70 mv
V FIB/V TACH
After phosphorylation/
phosphate cleavage
After Administration
Of Procainamide
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Use-dependent block of sodium channels.
Blocks potassium channels.
Blocks alpha-adrenergic receptors.
Blocks muscarinic receptors.
Used to attempt to terminate persistent reentrant
arrhythmias
• Reduces automaticity of ALL pacemakers (both
the SA node and ANY tissue capable of
generating a pacemaker potential)
• Slows Down Conduction of depolarization in ALL
tissues of the heart and decreases cardiac
excitability
• This is your last resort. Giving this drug may
stop the arrhythmia, but make it almost
impossible for the heart to spread impulses after
Summary For the Lecture
• Na+ channels are comprised subunits, the Alpha of 4
repeating motifs, each motif with 6 transmembrane
domains
• There are voltage, ligand, and mechanically-gated Na +
channels
• Na+ channels are involved in the depolarization of
excitable membranes
• Na+ channels have multiple modalities of modulation,
which can alter neuronal/membrane excitability
• Na+ channels are the target of a multitude of
pharmacological agents
Summary
• Na+ channels Are involved in the
remission-relapse of MS
• Na+ channel gating can be significantly
affected by modulation (phosphorylation,
mutation, proteolytic cleavage)
• Mutation in Nav 1.5 is implicated in Long
QT syndrome, generating persistent and
slow inactivating sodium current