Voltage-Gated Sodium Channels (NaV)

Voltage-Gated Sodium Channels (NaV) - Part 1

Overview

• The lecture focuses on the structure and function of voltage-gated sodium channels (NaV channels).
• Topics include: classification and gating of ion channels, NaV channel structure, voltage sensitivity, ion selectivity, inactivation, phosphorylation, and the effects of toxins and mutations on NaV channel function.

Major Neurotransmitter Systems and Ion Channel Families

• Major neurotransmitter systems include:
  • Glutamatergic
  • GABAergic
  • Cholinergic
  • Catecholamines (dopamine, noradrenaline, adrenaline)
  • Indolamines (5-HT)
• Major ion channel families:
  • Voltage-gated sodium channels (NaV)
  • Voltage-gated potassium channels (KV)
  • Voltage-gated calcium channels

Classification of Ion Channels

• Voltage-gated ion channels:
  • NaV channels
  • Voltage-gated calcium channels (VGCCs)
  • KV channels
  • Hyperpolarization-activated cyclic nucleotide-gated channels (HCN)
• Ligand-gated ion channels (ionotropic receptors):
  • Nicotinic acetylcholine receptors
  • Ionotropic glutamate receptors (AMPARs)
  • Acid-sensing ion channels (ASICs)
  • ATP-gated P2X receptors
  • GABAA receptors
  • Transient Receptor Potential (TRP) ion channels (e.g., capsaicin receptor TRPV1)
• Intracellular signaling messenger-gated channels:
  • Calcium-activated K+ channels (BK, SK, and IK channels)
  • Cyclic nucleotide-gated ion channels
    • CNG channels (cAMP and cGMP)
    • HCN channels (cAMP)
  • IP3 receptor (inositol trisphosphate)
• Other gating mechanisms:
  • Temperature-gated ion channels (TRPV1, heat; TRPM2, warm; TRPM8, cold)
  • Mechanosensitive ion channels (PIEZO channels)
  • Light-gated ion channels (channelrhodopsin or halorhodopsin)

Gating of Ion Channels

• Gating is the process of opening (activation) and closing (inactivation) ion channels in response to external signals.
• It is a rapid process, lasting about 1 msec.
• Gating can be influenced by:
  • Changes in membrane voltage
  • Binding of a ligand to the channel
  • Mechanical and physical forces
  • Changes in temperature
  • Activation by G-proteins
  • Catalytic modifications to channel

Ion Channel Selectivity

• Ion channel selectivity is determined by:
  • Size and charge of the ion
  • Spheres of hydration
  • Selectivity filters
• Example:
  • Potassium ions lose their spheres of hydration to fit through the pore.
  • Sodium ions retain their spheres of hydration and cannot pass through.

Active vs. Passive Conductance

• Active channels: Channels with gates that can open or close.
  • Voltage-gated ion channels
  • Ligand-gated ion channels
  • Temperature-gated ion channels
  • Mechanosensitive ion channels
• Passive channels (leakage channels): Channels that are always open.
  • Responsible for the resting membrane potential (RMP).
  • Example: K2P channels (background K+ channels)
  • KCNK 2-pore potassium channels mediate the passive leak conductance observed at rest.
  • If the RMP is depolarized, these open channels allow $K+$ to flow out of the neuron to repolarize the membrane potential (Vm) back to $E_K$.

Importance of Ion Channels

• Neurons cannot function without ion channels.
• Ion channels make neurons electrical.

Ion Channels and Electricity

• Membrane potential (Vm) is the voltage across the membrane at any moment, measured in millivolts (mV).
• At rest, $V_m = -65 mV$.
• The potential arises due to differences in electrical charge across the membrane.
• The inside of a cell is more negative relative to the outside.
• A negative charge inside of a neuron is essential for a functioning nervous system.

Resting Membrane Potential (RMP)

• The RMP is a balance between the equilibrium potentials for $K^+$ and $Na^+$, dependent on the membrane's permeability to each ion.
• $K^+$: inside = 100 mM, outside = 5 mM, $E_K = -80 mV$, permeability = high (40), some $K^+$ leaks out.
• $Na^+$: inside = 15 mM, outside = 150 mM, $E_{Na} = +62 mV$, permeability = very low (1), some $Na^+$ leaks in.
• Using the Goldman equation, the resting membrane potential is: $V_m = -65 mV$.

Ion Channels and Communication

• Ion channels allow neurons to communicate.

Action Potential

• The action potential has five phases:
  1. Resting state: The membrane is at the RMP ($-65 mV$).
  2. Rising phase: Rapid depolarization of Vm.
  3. Overshoot: The inside of the neuron is positive relative to the outside.
  4. Falling phase: Rapid repolarization of Vm.
  5. Undershoot: The inside of the neuron is more negative than at rest (hyperpolarized).

Channels Underlying Action Potential Phases

• Sodium (Na+) channels underlie the rising phase.
  • At threshold, voltage-gated sodium channels open and sodium flows into the neuron, depolarizing Vm.
• Potassium (K+) channels underlie the falling phase.
  • Voltage-gated sodium channels inactivate during the overshoot.
  • Voltage-gated potassium channels open and potassium leaves the neuron, repolarizing Vm.

Channel Kinetics and Action Potential Phases

• Synaptic potentials depolarize the neuron to threshold, activating NaV channels at the hillock.
• KV channels begin to open at the overshoot; NaV channels inactivate, and Vm approaches $E_{Na}$.
• Potassium flows out of the neuron through open KV channels, repolarizing the membrane potential; NaV channels are still inactivated.
• KV channels take longer to close, so Vm reaches $E_K$ producing the undershoot.
• During the undershoot, the Vm is refractory (absolute then relative).
• In the relative refractory period, NaV channels de-inactivate, allowing another action potential to be triggered.
• Different voltage-gated channels with unique kinetics and physiological properties underlie each phase of the action potential.

Voltage-Gated Sodium Channels (NaV Channels)

• The voltage-gated sodium channel is a large, multimeric complex composed of an alpha subunit and one or more smaller beta subunits.
• The ion-conducting aqueous pore is within the alpha subunit.
• The auxiliary beta subunits modify the kinetics and voltage-dependence of the channel's gating (opening and closing).

Activation of Voltage-Gated Na+ Channels

• Negative interior attracts the positive charge of the voltage sensor, holding the channel closed.
• A more positive interior repels the voltage sensor, opening the channel.
  • At threshold, the interior of the cell is positive enough to repel the positively-charged voltage sensor.
  • This causes the channel to change conformation, allowing $Na^+$ ions to pass through the pore.

Sodium Channel Currents

• Repeated depolarizing voltage pulses applied to the patch produce single-channel currents.
• Channels open most often in the initial 1 to 2 msec after the onset of the pulse, after which the probability of channel opening declines.

Sodium Channels and Inactivation

• Depolarization to threshold opens NaV channels.
• NaV channels inactivate a short time later (~1 msec).
• A portion of the channel blocks the open pore.
• Channels remain inactivated until repolarization to rest.

Process of Sodium Channel Inactivation

• A change in voltage across the membrane activates NaV channels, and sodium rushes into the neuron.
• This inward current is transient as NaV channels inactivate despite continued depolarization of Vm.
• NaV channels inactivate as Vm nears $E_{Na}$.

Types of NaV Channels

• Different NaV channels, their gene, location, and associated diseases:
  • Nav1.1 (SCN1A): Central and peripheral neurons, cardiac myocytes; Epilepsy, migraine, autism, Rasmussen's encephalitis, Lennox-Gastaut syndrome.
  • Nav1.2 (SCN2A): Central and peripheral neurons; Seizures, epilepsy, autism spectrum disorder.
  • Nav1.3 (SCN3A): Central and peripheral neurons, cardiac myocytes; Pain and epilepsy.
  • Nav1.4 (SCN4A): Skeletal muscle; Paralysis, myotonia.
  • Nav1.5 (SCN5A): Cardiac myocytes, skeletal muscle, gastrointestinal smooth muscle cells, central neuron; Long QT syndrome, idiopathic ventricular fibrillation, irritable bowel syndrome.
  • Nav1.6 (SCN8A): Central and peripheral neurons, heart and glial cells; Epilepsy.
  • Nav1.7 (SCN9A): Dorsal root ganglia, sympathetic neurons, Schwann cells, neuroendocrine cells; Erythromelalgia, channelopathy-associated insensitivity to pain, fibromyalgia.
  • Nav1.8 (SCN10A): Dorsal root ganglia; Pain, neuropsychiatric disorders.
  • Nav1.9 (SCN11A): Dorsal root ganglia; Pain.

Kinetic Properties of NaV Channels

• NaV channel subtypes (1.1 to 1.8) exhibit different kinetic properties and voltage dependencies.

NaV Channels Contributing to the Action Potential

• Nav1.6: Main contributor to the rising phase, rapid repriming supports high-frequency firing, high activation threshold.
• Nav1.7: Contributes to the rising phase and amplifies subthreshold stimuli, low activation threshold, fast kinetics, TTX-sensitive.
• Nav1.3: Contributes to the rising phase, rapid activation and fast inactivation, able to produce resurgent currents in some cells, TTX-sensitive.
• Nav1.8: Contributes to amplification of subthreshold stimuli when re-expressed after injury, low activation threshold, fast kinetics, TTX-sensitive.
• Nav1.9: Amplification of subthreshold stimuli, low activation threshold, ultra-slow kinetics, TTX-resistant.

Voltage-Gated Sodium Channels (NaV) - Part 2

Overview

• The lecture focuses on the structure and function of voltage-gated sodium channels (NaV channels).
• Topics include: classification and gating of ion channels, NaV channel structure, voltage sensitivity, ion selectivity, inactivation, phosphorylation, and the effects of toxins and mutations on NaV channel function.

Channel Kinetics and Action Potential Phases

• Synaptic potentials depolarize the neuron to threshold, activating NaV channels at the hillock.
• KV channels begin to open at the overshoot; NaV channels inactivate, and Vm approaches $E_{Na}$.
• Potassium flows out of the neuron through open KV channels, repolarizing the membrane potential; NaV channels are still inactivated.
• KV channels take longer to close, so Vm reaches $E_K$ producing the undershoot.
• During the undershoot, the Vm is refractory (absolute then relative).
• In the relative refractory period, NaV channels de-inactivate, allowing another action potential to be triggered.
• Different voltage-gated channels with unique kinetics and physiological properties underlie each phase of the action potential.

Types of NaV Channels

• Different NaV channels, their gene, location, and associated diseases:
  • Nav1.1 (SCN1A): Central and peripheral neurons, cardiac myocytes; Epilepsy, migraine, autism, Rasmussen's encephalitis, Lennox-Gastaut syndrome.
  • Nav1.2 (SCN2A): Central and peripheral neurons; Seizures, epilepsy, autism spectrum disorder.
  • Nav1.3 (SCN3A): Central and peripheral neurons, cardiac myocytes; Pain and epilepsy.
  • Nav1.4 (SCN4A): Skeletal muscle; Paralysis, myotonia.
  • Nav1.5 (SCN5A): Cardiac myocytes, skeletal muscle, gastrointestinal smooth muscle cells, central neuron; Long QT syndrome, idiopathic ventricular fibrillation, irritable bowel syndrome.
  • Nav1.6 (SCN8A): Central and peripheral neurons, heart and glial cells; Epilepsy.
  • Nav1.7 (SCN9A): Dorsal root ganglia, sympathetic neurons, Schwann cells, neuroendocrine cells; Erythromelalgia, channelopathy-associated insensitivity to pain, fibromyalgia.
  • Nav1.8 (SCN10A): Dorsal root ganglia; Pain, neuropsychiatric disorders.
  • Nav1.9 (SCN11A): Dorsal root ganglia; Pain.

Process of Sodium Channel Inactivation

• A change in voltage across the membrane activates NaV channels, and sodium rushes into the neuron.
• This inward current is transient as NaV channels inactivate despite continued depolarization of Vm.
• NaV channels inactivate as Vm nears $E_{Na}$.

Kinetic Properties of NaV Channels

• NaV channel subtypes (1.1 to 1.8) exhibit different kinetic properties and voltage dependencies.

Basic Structure of NaV Channels

• A functional NaV channel consists of a single alpha subunit (A). Accessory beta subunits regulate alpha subunit function.
• NaVs are monomers of around 2000 amino acids (single polypeptide chain).
• NaV channels consist of 4 separate domains (DI to DIV; A).
• Each domain consists of 6 transmembrane-spanning alpha helices (S1 to S6; A).
• Helices S1 to S4 make up the voltage-sensing domain (VSD), while S5 and S6 form part of the pore domain (PD) (A-C).
• This single polypeptide chain folds into a three-dimensional structure (B & C).
• When folded, NaVs look like they're composed of multiple subunits (tetramer-mimicking structure; B & C).

Inactivation Gate Location

• The portion of the channel responsible for inactivation is a sequence of amino acids that connect domains III and IV.

Voltage-Sensing Domain (S1 to S4)

• Alpha helices S1 to S4 make up the voltage-sensing domain (VSD), while S5 and S6 form part of the pore domain (PD).
• The charge distribution on S4 is critical.

Conserved Arginine Residues on S4

• There is a characteristic and conserved pattern in the location of arginine residues on S4 in both bacterial and eukaryotic NaVs.

Aligning Amino Acid Sequences

• Identifying regions that have been conserved over time is an important clue regarding the parts of a gene or protein of interest that are most important.

Arginine Repeats

• The arginine repeats every 3 amino acids on S4.
• This repetition ensures the residues align.
• Each arginine on S4 repeats every 3 amino acids, ensuring they're located at the same point along each bend in the coil.
• The arginine residues align at the same point on each bend in the coil of S4, forming a positively charged band along the length of the alpha helix.

Sliding Helix Model of Channel Activation

• S4 is pulled inwards when the neuron is at rest, but it is repelled outwards when Vm is depolarized.

S4 and Pore Domain Linkage

• S4 and S5 are connected to each other via a linker.
• Physical movement of S4 (courtesy of depolarization) pulls on the linker and shifts the position of S5 and S6 (change in conformation).
• Depolarization pushes S4 outwards.
• Because S4 is linked to S5, the outward movement of S4 shifts the lateral position of S5 and S6.

S4-S5 Linker

• The S4–S5 linker physically connects the voltage sensor to the pore domain and is essential for electromechanical coupling between them.

Lateral Bend in S5 & S6

• The arginine residues align at the same point on each bend in the coil of S4, forming a positively charged band along the length of the alpha helix.
• Physical movement of S4 (courtesy of depolarization) pulls on the linker and shifts the position of S5 and S6 (change in conformation), allowing the pore to open and sodium to enter.

Channel Gating in Action

• Simulations show the sliding helix model in action from three different views.
• S4 is repelled outwards, bending the S5 and S6 alpha helices laterally to change the conformation of the pore domain and open the channel.

Pore Domain & Selectivity Filter

• S5 and S6 make up the pore domain.
• Each of the 4 domains contributes S5 and S6 helices to form the functional pore.
• The pore consists of several regions, each contributing to ion conductance.

Selectivity Filter

• Four key residues form the selectivity filter, which are an aspartate-glutamate-lysine-alanine (DEKA) motif, consisting of one amino acid from each of the four pore loop regions from domains I to IV, respectively.
• Each domain contributes an amino acid to the selectivity filter.
• The residues allow the pore to be lined with negative side chains to attract $Na^+$ ions.

Voltage-Gated Sodium Channels (NaV) - Part 3

Overview

• The lecture focuses on the structure and function of voltage-gated sodium channels (NaV channels).
• Topics include: classification and gating of ion channels, NaV channel structure, voltage sensitivity, ion selectivity, inactivation, phosphorylation, and the effects of toxins and mutations on NaV channel function.

Lateral Bend in S5 & S6

• The arginine residues align at the same point on each bend in the coil of S4, forming a positively charged band along the length of the alpha helix.
• Physical movement of S4 (courtesy of depolarization) pulls on the linker and shifts the position of S5 and S6 (change in conformation), allowing the pore to open and sodium to enter.

Selectivity Filter

• Four key residues form the selectivity filter, which are an aspartate-glutamate-lysine-alanine (DEKA) motif, consisting of one amino acid from each of the four pore loop regions from domains I to IV, respectively.
• Each domain contributes an amino acid to the selectivity filter.
• The residues allow the pore to be lined with negative side chains to attract $Na^+$ ions.

Process of Sodium Channel Inactivation

• A change in voltage across the membrane activates NaV channels, and sodium rushes into the neuron.
• This inward current is transient as NaV channels inactivate despite continued depolarization of Vm.
• NaV channels inactivate as Vm nears $E_{Na}$.

Sodium Channel Inactivation Gate

• The hydrophobic IFM (isoleucine-phenylalanine-methionine) motif acts intracellularly as the gating particle that binds and occludes the pore during fast inactivation.
• Fast inactivation of NaV channels is crucial for preventing hyperexcitability.

Hinged-Lid Mechanism

• The IFM-motif acts as a hydrophobic latch that binds to sites on the S4-S5 linkers of DIII and DIV, as well as the cytoplasmic end of the DIV-S6 helix to close the activation gate from the inside.

IFM Function

• The IFM motif is required for NaV channel inactivation.
• Deletion or mutation of the IFM region prevents fast inactivation.

Beta Subunits

• NaV channels are comprised of one pore-forming α subunit and two non-pore-forming β subunits.
• The β subunits function as auxiliary subunits, which modulate the gating, kinetics, and localization of the ion channel pore.
• The five identified β subunits have also been shown to play many additional roles.
• The auxiliary beta subunits modify the kinetics and voltage-dependence of alpha subunit gating.
• For example, β1 and β2 increase the peak $Na^+$ current carried by NaV1.2, accelerate inactivation, and shift the voltage-dependence of activation and inactivation to more negative potentials.

Beta Subunits and Channel Kinetics

• β subunits (β1-β4) accelerate the activation and inactivation of NaV channels.
• The auxiliary beta subunits modify the kinetics and voltage-dependence of alpha subunit gating.

Phosphorylation of NaV Channels

• PKA (Protein Kinase A)
  • PKA phosphorylates NaV1.1 and NaV1.2 channels on four sites in the intracellular loop of domains I and II, causing a reduction in peak $Na^+$ currents.
  • However, phosphorylation of NaV1.8 by PKA increases $Na^+$ currents.
• PKC (Protein Kinase C)
  • PKC phosphorylates NaV channels, significantly slowing NaV1.1 and NaV1.2 channel inactivation (due to phosphorylation of a site in the inactivation gate) and reducing peak current (due to phosphorylation of sites in the intracellular loop between domains I and II).
  • However, phosphorylation of NaV1.7 & NaV1.8 by PKC increases $Na^+$ currents.

Pharmacology of NaV Channels

• Non-selective NaV blockers bind to the conserved residues in the S6 segments in domains I, III, and IV.
• Local anesthetics (e.g., lidocaine) and class I cardiac antiarrhythmics (e.g., flecainide) block NaV channels in sensory nerves (pain) and in the heart (arrhythmia).
• Anticonvulsants and antiepileptic drugs, such as carbamazepine and phenytoin, block NaV function.
• Antidepressants, like amitriptyline, also have effects on NaV channels.

Toxins

• Tetrodotoxin (TTX)
  • TTX inhibits sodium channels, producing heart failure and death.
  • TTX-sensitive (TTX-S): NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.6, and NaV1.7.
  • TTX-resistant (TTX-R): NaV1.5, NaV1.8, NaV1.9.
  • TTX binds to multiple sites in the pore-forming domain and blocks deep within the pore.

Fugu

• The origin of TTX is unknown, but in the pufferfish, it is produced by endosymbiotic bacteria.
• The ingestion of contaminated pufferfish is the usual route of toxicity.
• The restaurant preparation of “fugu” is strictly controlled by law, and only qualified chefs are allowed to prepare the fish.

Toxins Affecting NaV Function

• Pore-blocking toxins inhibit the flow of $Na^+$ by binding to the outer vestibule or inside the ion conduction pore.
• Gating-modifier toxins interact with a region of the channel that changes conformation during channel opening to alter the gating mechanism.
• Examples include: TTX, STX, μ-Conotoxins, Brevetoxins, Ciguatoxins, Scorpion α & β-toxins, Sea anemone toxins

Sodium Channels in Disease

• Inherited human disorders affecting skeletal muscle contraction, heart rhythm, and nervous system function have been traced to mutations in genes encoding voltage-gated sodium channels.

NaV Channels & Pain

• NaV1.7, NaV1.8, and NaV1.9 are expressed in peripheral DRG neurons mainly involved in the nociceptive pathway.
• Deleting NaV1.7 or NaV1.8 or NaV1.9 reduces inflammatory pain.
• Nonsense mutations in the NaV1.7 gene (SCN9A) caused a loss-of-function of NaV1.7 and a complete loss of ability to sense pain in patients.
• Gain-of-function mutations of NaV1.7 cause severe pain syndromes, such as inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD).
• Modification of NaV1.8 channels by methylglyoxal, a metabolite of glucose, causes hyperexcitability of DRG neurons and painful diabetic neuropathy.

NaV Channels & Epilepsy

• NaV1.1 and NaV1.2 channels are expressed in GABAergic interneurons.
• Loss-of-function mutations of NaV1.1 (20 mutations) and NaV1.2 (over 600 mutations) impair the excitability of GABAergic inhibitory neurons, leading to neuronal hyperexcitability and seizures.
• Anticonvulsants achieve their therapeutic effects by blocking NaV channels (e.g., phenytoin) or enhancing inactivation (e.g., lacosamide).

NaVs & Disease

• Cardiovascular Diseases
  • NaV1.5 is a major player.
  • Gain-of-function mutations in NaV1.5 prevent channel inactivation, generating persistent $Na^+$ currents, which underlies Long QT syndrome.
  • Loss-of-function mutations in NaV1.5 cause arrhythmia.
• Respiratory Disorders
  • NaV channel blockers (NaV1.7, NaV1.8, & NaV1.9) appear to work as effective antitussive agents.
• Cancer
  • In preclinical studies, inhibitors of NaVs inhibit proliferation, tumor growth, metastasis, and angiogenesis.

Channelopathies

• Normally, in response to a depolarizing voltage step, NaVs progress from the closed to open states and a large transient INaP is observed.
• Within a few milliseconds, the inactivation gate closes, and channels become inactivated.

Dysfunctional NaVs

• An epilepsy-causing channelopathy point mutation impairs inactivation of the NaV channel, leading to an increased INaP and excessive sodium influx at steady state, leading to hyperexcitability.