Voltage-Gated Ion Channels

Question 1

Link the structure to its function of voltage-gated Na+ channels

Answer 1

• These channels mediate the rising phase of an action potential.
• Transmembrane helix S4 of each of the 4 domains contain positively charged amino acids such as arginine and lysin. This allows it to act as a voltage sensor.
• Depolarisation-induced outward repulsion of s4 helices triggers the opening of VG Na+ channels.
• S5 and S6 make up the pore forming region of the alpha subunit, this allows for selective permeability.
• The intracellular loop linking domains 3 and 4 of the alpha subunit results in inactivation. In order to recover from this, channels must undergo deactivation where they return to their original closed conformation. This occurs when the membrane potential returns to its resting state level.
• Mutations of DEKA to either DEDA or DEKA converts sodium channels to a channel permeable to calcium ions.

Question 2

What are the different types of voltage-gated Ca2+ channels?

Answer 2

• These channels mediate neuronal excitation-secretion coupling by mediating entry if Ca2+ into the presynaptic terminal. They have the same features as VG Na+ channels.
  1. L-type (long lasting): High-voltage activation which are long lasting, blocked by dihydropyridines. These are involved in muscle contraction. All CaV-1 are L-types.
• dihydropyridines are allosteric modulators which alter gating behaviour
• Drugs acting on L types are normally used to treat hypertension or cardiac arrhythmias.
  2. N, P/Q, R-types: High voltage activation and are involved in NT release. These are not blocked by dihydropyridines.
• L types require large depolarisation (-20mV) to open and do not alway experience deactivation.
  3. T-types: Low voltage activation and are transient. They are not blocked by dihydropyridines. All CaV-3 are T-types
• Associated with the cardiovascular system.
• Transient types open at more negative values (-60) and experience rapid voltage dependent inactivation

Question 3

How do voltage-gated Ca2+ channels act as calcium sensors?

Answer 3

• At least one calcium ion is bound within the pore at all times, if the extracellular calcium concentration is low, then the channel becomes permeable to sodium.
• HVA channels are blocked via rise in intracellular calcium and is mediated by the calcium-binding protein calmodulin. As intracellular calcium levels rise this causes the channel to close and limits calcium signal duration.
• The glutamic acid residue (E) in the P region of each domain determines selectivity to Ca2+ ion.
• Ca2+ influx triggers fast evoked transmitter release.

Question 4

What are the properties of voltage-gated K+ channels?

Answer 4

• Mediate falling phase of an action potential as well as determines the rate of depolarisation.
• VG K+ channels also stop neurones from firing action potentials at low levels of depolarisation.
• S4 with positive amino acids for voltage sensitivity.
• Residues GYG that are part of the consensus sequence of K+ channels form the selectivity filter.
• Ball and chain mechanism of VG K+ channels causes inactivation in the manner that any of the 4 “balls” can block the pore.
• Na+ and Ca2+ channels are formed by the interactions of four different domains while K+ is the interactions between 4 identical subunits. They are formed via internal duplication.

Question 5

Explain the mechanisms behind the NaV channel.

Answer 5

• When the intracellular part of the cell becomes more positive, voltage gated channels are going to become more gated. This involves the movement of the S4 segment to the outside of the lipid bilayer. The channel opens.
• In response to depolarisation channels open, however if this depolarisation is maintained Na+ channel exits open state and enters inactivated state.
• Resting (closed) state is favoured by hyperpolarisation, open state is favoured by depolarisation and inactivated state is caused by maintained depolarisation.

Question 6

Describe the activity of NaV’s two gates.

Answer 6

• At resting membrane potential, the activation gate closes the channel.
• Depolarisation causes opening of activation gate allowing Na+ to enter the cell.
• The inactivation gate the closes stopping Na+ entry.
• Depolarisation caused by efflux of K+ causes the two gates to resume their original configuration.

Question 7

Pharmacology of NaV.

Answer 7

• 9 sodium channel alpha subunits in the human genome located in the CNS are sensitive to nM TTX. A toxin which blocks sodium channels causing muscle paralysis.
• However Nav 1.8 + 1.9 (expressed in dorsal root ganglion nerves) are resistant and are responsible for rapid firing of depolarised nerves. They play a vital role in chronic inflammatory pain and increase in proximal sites to any injuries.
• Local anaesthetics (Las) are small lipid soluble molecules which cross the nerve sheath and cell membrane to reach site of action (-caine).
• Las block Na+ channels in their open state (open channel block) and it blocks from the inside. The blockage is voltage dependent.

Question 8

What are some toxins which affect NaV?

Answer 8

• Tetrodotoxin (puffer fish) blocks channels from the outside. The toxin saitoxin (STX) has similar effects.
• A mutation in the channel P loop from glutamate (-E) to glutamine (Q) decreases TTX sensitivity reducing blockage, while cysteine (C) mutated to tyrosine (Y) increases effect.
• Mew-conotoxins (molluscs) are small positively charged peptides (specifically GIIIA) block skeletal Na+ channels.
• Batrachotoxin inhibits inactivation and shifts the activation voltage to more negative values thus prolonging duration.

Question 9

What are some toxins that affects CaV?

Answer 9

• Phenylalkylamine is an open channel blocker which binds to the inner end of transmembrane pore (site in s6).
• Benzothiazepines bind to residue in S5-6 linker in domain 4, both of these work as antagonists same as DHP.
• N, P/Q, and R are associated with the peripheral CNS therefore ziconotide which binds to N types deals with chronic neuropathic pain.
• Gabapentin is originally synthesised as GABA and is used to treat chronic pain via the A2 delta subunit.

Question 10

What are the three types of calcium activated K+ channels.

Answer 10

• Play a vital role in limiting Ca2+ entry and neuronal excitability.
  1. Large conductance K+ channels (Maxi-K channels).
  2. Intermediate (IK) conductance channels
  3. Small (SK) conductance channels.
• In neurons SK channels are responsible for the more persistent slow hyperpolarisation (AHP) observed after action potential discharges.

Question 11

Maxi-K Channels

Answer 11

• Expressed everywhere in neurons other then the heart. They help shape action potentials in neurons and regulate contractile activity + tone in smooth muscles.
• Voltage dependent (gated by depolarisation) , increasing Ca2+ concentrations allow channel opening, the S4 domain acts as the voltage sensor.
• Has both a ligand gating and voltage gating domain, 2 independent sensing mechanisms with a low probability of channel being open when neither are activated.
• Acts as a negative feedback mechanism for calcium entry, also contributes to after-hyperpolarisation (AHP). Also works to balance excessive vasoconstriction

Question 12

What are some specific uses of Maxi-K?

Answer 12

• When either Ca release by CICR via ryanodine receptors or calcium entry via CaV. It causes a STOC spontaneous transient outward current so a Ca2+ efflux.