PCOL3022 Lecture 2: VGICs

Resting Membrane Potential (functional state of voltage dependent ion channels)

- -70mV
- More negative inside
- Na+ ions outside and K+ inside
- Na+ and K+ channels are closed

Where is the selectivity filter in VGICs?

In the extracellular region of the channel

A drug that modulates the function of an accessory subunit

Phosphorylation of beta subunits can regulate VGICs function

Compare the structures K+, Na, Ca2+ and TRP channels.

- Na+ contains internal homologous 4 subunits and 6 transmembrane domains. S4 contains positive residues (voltage sensor). S4-S5 is the linker domain.
-Ca2+ has a similar structure to Na+ VGICs
- K+ VGICs contains 4 subunits but only one of the subunit correspond to the internal homologous of Na+ channels
- TRP contains similar structure (i.e., 4 subunits x 6 transmembrane) but lack positive residues in S4.

How do local anesthetics inhibit Nav? Identify different channel states and modes of access to the channel.

- E.g. cocaine, procaine, lidocaine

- LAs bind to the fenestrations in the pore of the Nav channels

- Causes Na+ to bind to the inactive state hence blocking the channel

Pain Treatment in Nav channels

- Mutations to Nav1.7 cause inability to sense pain
- Potential drug target
- Requires high selectivity to prevent fatal effects from blocking related Nav channels

- Potential selective inhibitors include:
> PF-05089771
> Monoclonal antibodies
> Tarantula toxins

What is an action potential?

An electrical signal that triggers the release of NTs throughout the axon, further relaying the signals.

What are K+ channels located?

Dendrites

What are Ca+ and Na+ channels mainly located?

Presynaptic nerve terminals and axon shaft (i.e., axon hillock and node of Ranvier)

How is ion selectivity achieved?

There is a selectivity filter deep inside the channel. In this narrow opening, residues select specific cations which allows for permeation of specific ions

What are the different types of VGICs?

Na+, Ca2+, K+, TRP

What is the structure of VGICs and the key features?

- Na+ contains internal homologous 4 subunits and 6 transmembrane. S4 contains positive residues (voltage sensor). S4-S5 is the linker domain.
-Ca2+ has a similar structure to Na+ VGICs
- K+ VGICs contains 4 subunits but only one of the subunit correspond to the internal homologous of Na+ channels
- TRP contains similar structure (i.e., 4 subunits x 6 transmembrane) but lack positive residues in S4.

Na+ channel structure and key features

- Contains internal homologous 4 subunits and 6 transmembrane domains
- S4 contains positive residues (voltage sensor)
- S4-S5 is the linker domain

Ca2+ channel structure and key features

- Has a similar structure to Na+ VGICs

K+ channel structure and key features

- Contains 4 subunits
- But only one of the subunits corresponds to the internal homologous of Na+ channels

TRP structure and key features

- Contains 4 subunits x 6 transmembrane domains
- Lacks positive residues in S4

Which VGIC has more positive charges on the S4 domain, Na+ or K+?

Na+

What are the different functional states of VGICs?

Open state
VGICs activate in response to depolarization. Voltage sensory (SR) tilts 25 degrees and 3A away from the resting state. This causes the S4-5 linker to pull outwards and open the pore.

The ligand can also bind to regulatory domains which regulate opening channels. For example, Ca2+ binding can activate and open Ca+ activated k+ channels.

Close state
Deactivation state: VGICs return back to the resting membrane (opposite of activation)

Inactivation state: referring to the ball and chain model, the ball blocks the pores and prevents ions from flowing in

Can alpha accessory subunits function on their own?

Yes

What pulls the pore open?

S4-S5 linker

How does local anaesthetics affect Nav?

Local anaesthetics can bind to sodium channels to rapidly block nerves and reduce undesirable reactions such as pain.

How does tetrodotoxins affect NaV channels?

- Tetrodotoxins are NaV blockers and are found in marine species

- Binds external surface of alpha-subunit in S5/S6 loop region (which blocks the pore)

- Variations in this region generate channel selectivity

Note: only used in vitro, experimentally

How does Phenytoin and Carbamazepine affect NaV channels?

- Treats epilepsy (preventing seizures)

- Ph and Carb slow down the recovery from inactivation state => limiting firing rates

What are the two classes of K+ channels? Provide examples.

6TMK+: K+ VGICs and Ca+ activated K+ channels
2TMK+: inwardly rectifying K_ channels

What are the two functional groups of K+ channels, and elaborate on their functions?

Delayed rectifying: delayed activation of K+ channels after depolarisation and inactive slowly => facilitate repolarisation

A type: k+ channels undergo rapid activation and inactivation to reduce neurons firing frequency

What are the diverse functions of CaV channels?

- Apoptosis
- Muscle contraction
- NTs release at presynaptic neurons
- Activates 2nd messenger

How does tetraethylammonium block Kv channels?

TEA mimics K+ ions and blocks most types of K+ channels.

What can calcium channels trigger?

Ca+ triggers apoptosis, muscle contraction, release of NTs in presynaptic cleft and 2nd messenger activation

How does gabapentin affect Cav channels?

It ↓ excitatory transmission by acting on voltage-gated Ca²⁺ channels presynaptically (α₂ δ subunit).
(Does not bind GABAa/b)

When are A-type Kv channels activated and what are their roles?

A type: K+ channels rapidly activate and inactivate allowing for reduced neuron firing frequency

What is the function of Ca2+ activated K+ channels?

Ca2+ ligands can bind and activate Ca+ activated K+ channels

What are the functions of Cav channels?

CaV involves in apoptosis, muscle contraction, NTs release at presynaptic terminals and activated 2nd messenger

What are the Cav channel subtypes?

CaV1.1 = L type
CaV1.2 = L type
CaV2.1-2.3 = P/Q N and R type
CaV3.1-3.3 = T type

What are the functions of Cav channel subtypes?

CaV1.1 = L type = activate skeletal muscles
CaV1.2 = L type = activate skeletal, smooth and cardiac muscles
CaV2.1-2.3 = P/R N and Q type = influx of Ca2+ allows for the release of NTs (note: blocking CaV2.2. used in chronic pain treatment)
CaV3.1-3.3 = T type = repetitive firing of neurons

What do TRP channels do?

Detects pain, temperature, touch and taste

Which TRP channel for pain perceptions and chemical/physical stimuli?

Pain perceptions: TRPV1 and TRPV1
Chemical/ physical stimuli: TRPV1

How do TRP channels work?

- Nociceptive pain activates ion channels in dorsal root ganglion neurons leads to Na+ and Ca2+ influx

- Causes membrane depolarisation which triggers VGIC-dependent action potential to send information to spinal cord and higher nerve centres

Can TRP channels be activated by something else? Elaborate

TRP can be activated by ligands such as capsaicin, chilli and cannabinoids.

Depolarisation (functional state of voltage-dependent ion channels)

- +30mV
- Becomes positive inside
- Na+ VGICs open => Influx of Na+ inside

Repolarisation (functional state of voltage-dependent ion channels)

- Making inside less positive
- Na+ channel is inactivated
- K+ VGICs open => Efflux of K+ to outside
- Sodium and potassium pump => 3 Na+ out and 2 K+ in

Hyperpolarisation (functional state of voltage-dependent ion channels)

- Very negative inside
- Na+ VGICs closed
- Pump + K+ VGICs open => hyperpolarized

Relative Refractory period (functional state of voltage-dependent ion channels)

- K+ channels close => resting membrane potential
- Only strong stimuli can activate AP again

What selects different cations in selectivity filters of VGICs

In this narrow opening, there are specific residues that select different cations

Alpha subunit function

Alpha subunits can function on their own

What subunit does Beta subunit regulate?

Alpha subunit

Beta subunit function

- Involves in expression levels, location (i.e., where the channel is expressed)
- Alters voltage-dependent (manipulate the sensitivity of activation and deactivation) trafficking

What is Na+/K+ ATPase?

- Energy derived from hydrolysis of ATP

Location of VGICs

- Throughout the neuron
- Localised to specific locations (Nav, KCNQ in AIS)

Which LGIC doesn't have all the positive residues in S4 domain?

TRP

Kv structure

- Each single subunit is a separate protein
- These tetramerise to form the channel
- Allows huge diversity of K channels
- Some K channels exist as only the S5 and S6 domains (these line the pore)

Nav, Cav structure

- 4 alpha subunits
= Each subunit has 6 TMDs (S1-S6)
= S4 is full of positive residues

- 2 beta accessory subunits
= These allow modulation of function

Role of Accessory Subunits

- B subunits are diverse and modulate the function of the alpha subunit

1. Can regulate expression levels, location, trafficking
2. Can alter voltage-dependence of activation/inactivation
3.Can bind drugs that modulate
4. Can regulate VGIC functions via phosphorylation

Molecular properties of VGICs

- the aqueous pore control selectivity
- all VGICs have similar pore structures

Which VGIC is the most selective?

- Ca2+ channels = 1000-fold more selective over other cations

How does VGIC selectivity work?

- Aqueous channel itself has selectivity
- Selectivity filter is lined with differently charged residues that confer selectivity
- Aqueous pore can close to prevent passage of ions

Open Functional State of VGICs

The charged S4 TMD (one with positive residues) will move upon a change in membrane potential

= Shifts away from more positive side and towards less positive side
= Total change ~3 A and 25 degrees
= This shifts a linker protein that pulls the protein open

Closed Functional State of VGICs

1. Deactivation
> Fast
> Once the change in the membrane is reversed, the S4 moves back, and the channel closes
> VGICs return back to the resting membrane (opposite of activation)

2. Inactivation
> Slow
> Nav channels close immediately after activation
> Influx of positive charge causes intracellular domain (ball) to swing via "chain" into the open pore, effectively plugging the pore
> Ball blocks the pores and prevents ions from flowing inside
> Referred to as the "ball and chain" model

What happens when Nav channels are blocked?

- Blocking all Nav channels is very dangerous
> Loss of all sensory system, sedation, coma
> Only done in vitro to study other channels in isolation

Nav Channels Modulation

- Selective modulation allows therapeutic effect

- Many Nav blockers can only bind open state (i.e. active neurons)
> Rates of (in)activation therefore allow selectivity

- 9 major types of alpha-subunit with different tissue distributions also allow selectivity

K+ channels functions

- K+ channels are a stabilising force:
> Sets cell's resting membrane potential
> Repolarizes cell after action potential
> Controls rate of firing and shape of action potential

K+ channels TMDs

- Some channels have 6TMDs
- Some have 2TMDs (S5 & S6 only)
> The 2TMD channels are NOT voltage-dependent

K+ Channels Types

- Differences are with how the gate receives signal, structures are similar

1. Delayed rectifiers
> Delayed activation after depolarisation, inactivate slowly --> facilitate repolarisation

2. A-type channels
> Transiently activated when cell is depolarised after hyperpolarisation --> decreases firing frequency

3. Ca2+-activated K+ chanels
> Respond to Ca2+, stays open for long periods --> prolong hyperpolarisation

4. Inward rectifiers
> Activity regulated by G-proteins (GIRKS)

Kca channels

- Regulate neuronal and smooth muscle excitabilities
- Open when Ca2+ binds after depolarisation allows Ca2+ entry
- Remain entry for a few seconds
- Causes "long after hyperpolarisation" i.e. hyperpolarisation after an action potential --> slows firing rate of action potentials

Functions of Ca2+ channels: L-type

1. Skeletal muscle (1.1)
2. Cardiac and smooth muscle, brain (1.2)
3. Channels found in cell body and proximal dendrite
4. Inhibited by verapamil, nifedipine

Functions of Ca2+ channels: P/Q, N and R type

1. Regulation of NT release (Ca2+ influx via channels prompts release)
2. Potential drugs for treating chronic pain -- synthetic peptide that is an N-type channel blockFunctions of Ca2+ channels: L-type

Functions of Ca2+ channels: T-type

1. Repetitive firing of neurons