Ion channels L4

Classify ion channels based on their structure and gating mechanism

• open/leakage channels - 4 subunits, 2 helices, controlled by structure of channel

• voltage gated - 4 subunits, 6 to 24 helices, p loop, cytoplasmic anchors, voltage sensing domains, plugging mechanism

• ligand gated, both IC and EC - 4 subunits, 6 helices, cytoplasmic anchors

Explain plugging mechanism in voltage-gated ion channels

intracellular portion of the channel acting as a "plug" or "ball and chain", blocks the pore shortly after it opens. rapid closure, even when the membrane is still depolarized, stops ion flow and is crucial for the channel's function and the generation of action potentials by creating a refractory period

Identify features and function of ligand-gated ion channels.

excitability, signalling, secretion, absorption, and muscle function

Explain the structural basis of ion selectivity and describe how pore architecture and amino acids determine which ions pass through

• pore architecture: selectivity filter, narrow region of the pore that matches the size and charge of the target ion, only ions that can fit and shed their hydration shell correctly can pass

• amino acids: Carbonyl oxygens or charged residues line the filter, their spacing mimics the ion’s normal hydration envt, stabilising the right ion but not others

Compare and contrast key features of different ion channels

Describe how dysfunction of ion channels can contribute to disease.

Mutations or misregulation of ion channels contribute to conditions such as epilepsy, stroke, ALS, glioblastoma, and nicotine addiction.

Evaluate how ion channels serve as drug targets

Ion channels are attractive drug targets because of their diversity and specificity, physiological importance, and role in disease.

What is a transmembrane protein?

protein channel that transports molecules from one side of the membrane to the other

What are ion channels specific to?

open or gated, specific to sodium, potassium, chloride

What are essential functions carried out by ion channels?

• transport ions across membrane (secretion/absorption of fluids)

• regulate membrane potentials (nerve + muscle cells for high-speed communication)

• calcium influx into the cytoplasm (secretion + muscle contraction)

Name examples of areas where ion channels transport fluids/ions across membranes

salivary glands and kidneys

Name a way in which ion channels can regulate membrane potentials. Where would these channels therefore most likely be found and why?

open channels = ions to diffuse down the concentration gradients into the cell so can control the membrane potential = electrical signal that spreads very rapidly over the cell surface

nerve and muscles cells use these action potentials for high speed communication

Name a way in which ion channels allow secretion and muscle contraction

Ca2+ influx into the cytoplasm from ER or outside the cell where it can be used for a number of processes such as secretion or muscle contraction.

What are common structural features to all ion channels?

• transmembrane, two or more alpha helices crossing lipid bilayer

• 2 - 6 subunits surrounding a pore

What structural differences are ion channel subgroups based on?

• Gating mechanism

• Ion Selectivity of the pore

What defines the ion selectivity of a pore?

by physical size of ‘filter’ and amino acids lining the pore

How many genes in humans code for membrane channels?

400

Considering that structure of ion channels has revealed evolutionary relationships between ion channels, what can the KscA potassium channel found in bacterium be used for?

serves as a model for all channels

Describe the molecular structure of a simple potassium ion channel like the KcsA channel

highly selective TM helicase structure forming a p - loop (pore)

on cytoplasmic side, TMs more tightly packed = gate

What 3 factors control a gate on a simple ion like potassium’s channel?

• membrane potential

• mechanical stress

• ligands

What are the 2 main functions of voltage gated ion channels?

• sodium and potassium create action potentials in excitable cells 

• calcium is transported into cytoplasm where 2nd messenger = cellular response 

What distinguishes a voltage gated ion channel from a simple ion channel?

• Additional helices S1 and S4 form a separate ‘voltage sensing domain’ lateral to the subunits

• Large polypeptides that extend into the cytoplasm

• Plugging mechanism

Explain how this voltage gated potassium ion channel opens/closes

• Voltage-gated K⁺ channels have 4 subunits, each with 6 transmembrane helices (S1–S6).

• S4 helix, rich in positively charged residues, senses changes in membrane potential.

• Depolarisation → S4 moves outward → S4–S5 linker pulls open S6 → channel opens.

• Repolarisation → S4 returns inward → S6 closes → channel closes.

Describe the ion channel seen on this image

Voltage gated potassium ion channel: 

• The ribbon model shows two of four subunits of a Kv channel.

• Each subunit has six transmembrane helices (S1–S6).

• S1–S4 make up the voltage-sensing domain (VSD).

• S5–S6 form the pore domain, including the selectivity filter and activation gate.

• The S4 helix (positively charged) is key to sensing voltage changes.

Describe the resting state of a voltage gated potassium ion channel

• The inside of the cell is negatively charged relative to the outside.

• The positively charged S4 segment is attracted inward (toward the cytoplasm).

• This keeps the activation gate (at the S6 helices) closed, preventing K⁺ flow.

Describe a voltage gated potassium ion channel when depolarising/activating

membrane potential becomes less negative:

• The reduction in negative potential repels the S4 segment’s positive charges, causing it to move outward (toward the extracellular side).

• This movement pulls on the S4–S5 linker, which in turn opens the activation gate in the S6 region → K⁺ ions flow out.

Describe a voltage gated potassium ion channel when repolarising/inactivating

• When the membrane potential returns to negative, S4 moves back inward, releasing tension on S4–S5.

• This allows the S6 helices to close again, blocking the pore.

• Some Kv channels also have a “ball-and-chain” inactivation mechanism, where a polypeptide segment (often from the N-terminus, not S4) transiently plugs the pore.

How are ligand-gated ion channels similar and different from voltage-gated ion channels? 

similar structure, just controlled by different factors, former by the binding of a ligand 

Are ligand-gated channels controlled by IC or EC ligands? Name examples of these types of channels?

can be either ! IC is eg Cyclic nucleotide ligand, Ec is eg nicotine, glutamate, ATP

How many subunits and helices go across the lipid bilayer in simple KcsA, voltage-gated and ligand-gated channels?

4 subunits for all, 2 helices for KcsA , 6 to 24 for voltage-gated and 6 for ligand-gated

Which types of ion channels have a p loop, a cytoplasmic anchor, a voltage sensing domain/a plugging mechanism?

voltage-gated has all, ligand-gated has p loop and cytoplasmic anchors and KcsA just has the p-loop

What cellular process are extracellular ligand-gated ion channels important in?

cell-cell communication

What are distinct families of extracellular ligand-gated receptors? Are they tri/tetra/penta - metric?

• ATP 2X : trimeric P2X 1 to 7

• glutamate: tetrameric AMPA, NMDA, KA

• nicotinic receptor superfamily: Pentameric nAChRs, 5-HTR, GABAaRs, GlyRs, GluCl, MOD-1

Name some specific functions of channels selective for sodium or potassium

control membrane excitability – depolarize cells 

Name some specific functions of channels with added permeability to calcium

directly regulate activity of calcium sensitive proteins

Name some specific functions of channels that are chloride selective

control membrane excitability – reduce resistance/ hyperpolarize cells, reduce action potential firing

In the specific example of a cys-loop type receptor like the nAChR, describe its structure in muscle

• 5 subunits (alpha, beta, gamma, epsilon)

• each subunit has 4 TM M1, M2, M3, M4 with

• a large external facing N domain and IC loop between M3 and M4.

• M2 lines the pore.

• have small IC domains anchoring them into the cell membrane

In the specific example of a cys-loop type receptor like the nAChR, describe its mode of action towards muscle contraction

NT binds - opens channel - non selective flux of cations - electrical change = muscle contraction

How is added complexity added into ligand-gated ion channel families? How does this affect drug targeting?

different subunit combinations make up receptors in different parts of the brain - complexity = diversity ie opportunity for specific drug targeting 

Which ligand-gated ion channel is involved in reward pathways and nicotine addiction? How could it differ from other channels within the same ligand-gated ion channel family?

nACh alpha4 - different subunits to others within same family like nAChalpha 1, 2, 3, 7, 9, 10 etc

Which subunit variations define nAChR affintiy for nicotine? What else could affect this?

alpha 2 to 10 and beta 2 to 4 - huge amount of variability between these receptors depending on subunit associations

composition and location of receptors and subunits

Which type of receptor is abundantly expressed in the cortex and hippocampus and have high affinity to agonists nicotine and varenicline?

nACh alpha 4 beta 2

What does chronic exposure of nicotine receptors to agonist lead to?

receptor upregulation ie more nicotine = more receptors made = more addiction

When conducting genetic studies on nAChRs, what polymorphisms in subunit genes were found to be linked to tobacco dependence? What does this knowledge allow?

What other variants have been found that could allow for treatment against tobacco dependence?

subunit genes CHRNA$ (alpha 4) and CHRNA6 (alpha 6) - linked to tobacco dependence

variants shown to be protective against dependence - could be exploited for treatment?

What causes autosomal dominant nocturnal frontal lobe epilepsy? How many mutations have been identified causing ADNFLE?

mutation in the M2 region of alpha 4 neuronal nicotinic subunit so in nAChR

9!

How do mutations in M2 region of alpha 4 neuronal nicotinic subunit affect nAChR function and cause ADNFLE?

M2 mutations in α4 subunit alter the ion pore → increase receptor open probability and slow desensitization → enhance nicotinic signalling → cortical hyperexcitability → ADNFLE seizures.

How does a sustained depolarised nACh M2 mutated receptor lead to ADNFLE?

• Mutant α4β2 nAChRs in thalamocortical neurons become overactive.

• excessive ACh-induced depolarisation and enhanced transmitter release.

• The neuronal network becomes hyperexcitable, especially during non-REM sleep, when ACh levels fluctuate.

• This leads to seizure activity localized to the frontal lobe, producing nocturnal motor seizures.

Which is the main NT in the brain? Briefly describe the structure of the receptor to which this NT binds

glutamate

• glutamate receptors are tetramers similar to KcsA but the pore is inverted

• form as dimer of dimers, ligand binding site is in a cleft that closes when occupied

What contributes to glutamate receptor diversity? Name examples of these receptors

Give some examples of glutamate Kainate receptors

GluK 1 - 5

Name some examples of glutamate NMDA receptors

GluN1, GluN2 A- D

Name examples of glutamate AMPA receptors

GluA1 - 4

What is the role of AMPA receptors?

mediate fast excitatory synaptic transmission in the central nervous system

What is the role of NMDA receptors?

N-methyl-D-aspartate receptor – involved in learning and memory – slower than other isoforms

What is the role of kainate receptors?

similar to AMPA but lesser role at synapses linked to Schizophrenia, depression and Huntingtons

What are 2 types of RNA processing that can affect receptor subunits?

RNA splicing and editing

How do the kinetic properties of flip differ from those of flop in AMPA receptors? 

flop is desensitised  at a quicker rate and has a reduced current response to glutamate than flip

How does the presence of isoforms of AMPA receptors throughout different regions of the brain allow for tuning of synaptic responses and plasticity?

Different brain regions express different ratios of flip/flop isoforms = tuned synaptic responses and plasticity ie how long AMPA receptors stay active after glutamate release

What are the two isoforms under which each subunit of AMPA receptors exist?

2 splicing isoforms of the subunit genes like GluA 1-4 - flip and flop, basically EC ligand binding loop of the proteins is different due to alternative splicing of 2 exons in the primary transcript 

In an AMPA receptor like GluA2, where is the Q/R site located and what is its function?

located in the M2 of the subunit, inside the channel pore

RNA editing site

How does the Q/R site of an AMPA receptor carry out RNA editing?

RNA editing enzyme ADAR2 converts the genomically encoded CAG (glutamine, Q) into a CGG at the RNA level = CGG (arginine, R) in the protein ie a Q to R site where rna is edited from original dna code

How do functions of unedited Q RNA differ from edited R RNA in AMPA receptors?

unedited allows calcium through the AMPA receptor so has a high permeability to it // edited blocks calcium entry so has a low calcium permeability

What does the edited R form of AMPA receptors protect neurons from, considering their permeability to calcium? Are there more edited or unedited GluA2 subunits in the brain’s AMPA receptors? 

excitotoxicity 

more edited ie R form - makes AMPA receptors largely impermeable to calcium, maintaining neuronal stability 

What happens in cases of failure for RNA to be edited at the Q/A site of an AMPA receptor? What could cause this type of failure?

AMPA receptors would be calcium permeable in their Q form = too much calcium = neuronal hyperexcitability, seizures and cell death

loss of ADAR2 enzyme or ADAR2 knock-out

Considering the speed of desensitisation of flip isoform AMPA receptors, what type of synapses are these receptors mostly involved in? How fast is their recovery time and what type of response do they have to glutamate?

slow desensitisation so involved in synaptic plasticity allowing learning and memory - fast recovery and sustained response to glutamate

Considering the speed of desensitisation of flop isoform AMPA receptors, what type of synapses are these receptors mostly involved in? How fast is their recovery time and what type of response do they have to glutamate?

fast desensitisation so involved in rapid synapses like high-frequency transmission and precise timing often found in auditory circuits

slow recovery and short responses to glutamate

Where would there be a higher flip to flop ratio of AMPA receptors in the brain?

hippocampus 

Where would there be a higher flop to flip ratio of AMPA receptors in the brain?

cerebellum and auditory neurons

What could a pathologically high expression of flip AMPA receptors lead to?

longer activation and slower desensitisation = Hyperexcitability, risk of seizures or excitotoxicity

What could a pathologically high expression of flop AMPA receptors lead to?

rapid desensitisation and reduced glutamate response = synaptic weakening, potential cognitive deficits

Consequence of AMPA dysfunction 

calcium permeability of AMPA = seizures and death 

Consequence of NMDA dysfunction

stroke and neuron death

What is thought to be the role of NMDA receptors?

controlling synaptic plasticity and mediating learning and memory functions

In glioblastoma, decreased ADAR2 activity correlated with increased malignancy bc of an increase in Ca²⁺ = Akt pathway promoting proliferation and tumorigenesis. What is therefore a potential therapeutic application?

GluA2 Q/R was editing = increasing survival bc increased nb of edited AMPA receptors than unedited reduced the calcium and the Akt pathway, reducing proliferation of tumours

Name some pathological conditions in which glutamate receptor RNA editing is involved 

forebrain ischemia, amiotrophic lateral sclerosis (both GluA2 receptor subunit), spinal cord injury (GluA2, 3, 4 and GluK1, 2), excitotoxicity, epilepsy, schizophrenia, bipolar disorders, antidepressant treatments, glioblastoma, fear conditioningm al

What type of ligand binds to P2X receptors? Briefly describe its structure

adenosine triphosphate (ATP)

trimeric assembly, 3 subunits with 2 TM helices, large EC domain

How many molecules are needed to open a P2X channel?

3 ATP molecules needed to open channel

What types of subtypes of subunits do P2X receptors have?

P2X 1 to 7

Consequence of P2XR dysfunction 

• microglia: modified stimulation of BDNF synthesis in microglia - depolarising shift in the anion reversal potential underlying neuropathic pain

• macrophages: modified activation of the caspase 1 inflammasome, release of inflammatory cytokines

• neurons: activation in stroke induces excitotoxic neuronal cell death by calcium overload. P2X 7 blockade = reduced brain tissue damage after tMCAO

Define gating

conformational mechanism by which ion channels open or close in response to voltage, ligand binding, or mechanical force. It allows neurons to control ion flow precisely, enabling electrical signalling, synaptic transmission, and sensory responses.

What causes ion channel diversity?

multiple subunit isoforms, RNA editing, alternative splicing