Sodium Channel Structure, Function, Gating and Involvement in Disease

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Sodium Channel Structure, Function, Gating and
Involvement in Disease
David R. Marks, M.Sc.
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An Overview

• Sodium Channel Structure
• - Current theory and Types of Na Channels
• Sodium Channel Function
• Current theory of inactivation
• Modulation
• Pharmacology
• Activation

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An Overview Contd

• Article 2 Na Channel Gating
• Article 1 - Na Channels and Neurodegenerative
  Disease
• Article 3 Na channel mutation and physiology

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Sodium Channels - Structure

• Composed of a, ß-1 and ß-2 subunits, but the
  large a-subunits 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

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Cartoon representation of the typical
voltage-activated sodium channel
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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

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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

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The theory is that the inactivation gate swings
shut, turning off the channel
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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

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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

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Why Na Channels/Modulation Are Important

• 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

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Na Channel Blockers/Pharmacological Agents

• Tetrodotoxin (TTX)
• Amioderone
• Lidocaine
• Procainamide
• Mexilitine
• Ketamine
• Many, many others

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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

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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

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Patch Clamping/Transfection
Transfection 1. Kv1.3 cDNA in
Plasmid 2. Lipofectamine complexing 3. Add to
Dishes 4. Patch 28-48 hrs after
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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

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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
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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

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Mutation alters the biophysical properties of the
channel
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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
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Other mutations to the Na channel Do not exert
as significant effects in the activation (V ½)
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Influence of hybrid Na channel subunits on
gating and biophysical properties
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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

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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 (gt
10µm)
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B-amyloid are pepties associated with
neurodegenerative diseases, and can accumulate in
fibrillar aggregates
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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

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An increase in NaV1.6 yields an Increase in
Na/Ca exchangers, elevating intracellular Ca2 to
harmful levels
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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

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Article 3

• Authors generated a similar mutant by
  site-directed mutagenesis
• Examined the mutant in a heterologous expression
  system to obtain biophysical and other properties

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The Nav 1.5 V1763M mutant is Sensitive to TTX,
but resistant to lidocaine
TTX eliminates lidocaine-insensitive current
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Why this is important Other than traumatic
cardiac arrest, arrhythmias degenerate into
ventricular fibrillation or ventricular
tachycardias. circus movement whereby tissue
becomes hyper-excitable
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Extension and Application of Na Channel
Properties and Function Relating to Article 3
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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
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40 mv
-70 mv
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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

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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

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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