L4 - Ligand gated ion channels

how are cells able to respond specifically to particular stimuli?

they have specific receptors and signalling molecules that control how they react

what is a channel?

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

• they are specific (Na+, K+ or Cl- ions, open or gated)

what are the essential functions of ion channels?

1. transport ions across the membrane

   (through secretion or absorption of fluid)

2. regulate membrane potential

   (expressed in nerve/muscle cells for high speed communication)

3. Ca2+ influx into the cytoplasm - transporting calcium

   (secretion + muscle contraction)

what are the structural features of ion channels?

• transmembrane proteins made up of two/more alpha-helices that cross the lipid bilayer

• they are composed of 2-6 subunits - each containing two/more alpha-helices

• these subunits surround the pore

what are ion channels classified into subgroups based on?

1. gating mechanism

2. ion selectivity of the pore

what is ion selectivity of the pore determined by?

the physical size of the pore’s filter and the amino acids lining the pore

what is the structure of a simple ion (K+) channel?

• transmembrane a-helices from each subunit form a p-loop (pore loop)

• the p-loop forms the selectivity filter

• the selectivity filter is highly selective for K+ ions

• on the cytoplasmic side the TMs are more tightly packed forming a gate which controls ion flow

what are the factors that control the gate (opening/closing)?

1. membrane potential

2. mechanical stress

3. ligand binding

what are the two man function of voltage gated ion channels?

1. Na+ and K+ channels = responsible for creating action potentials in excitable cells

2. Ca2+ channels = allow calcium ions to be transported into the cytoplasm → 2nd messengers elicit a cellular response

what is the structure of voltage gated ion channels?

(similar structure to simple ion channels)

• except of the additional helices S1 and S4 which form a voltage-sensing domain lateral to the subunits

• large polypeptides that extend into the cytoplasm

• plugging mechanism (can close the gate)

what does the voltage sensing domain do?

• senses changes in the membrane potential and triggers channel opening

what is the similarity and difference between voltage gated ion channels and transcient receptor potential (TRP) channels?

• they have common structural features to each other

• however TRP channels are evolved to sense chemical and physical stimuli

Intracellular ligand gated ion channels

• activated by ligands that bind inside the cell (cytoplasmic domain) or by intracellular second messengers

extracellular ligand gated ion channels

• Activated by ligands (e.g., neurotransmitters) binding outside the cell on the channel’s extracellular domain

what are extracellular ligand gated ion channels classified as?

• they are classified as a type of ionotropic receptor

what are types of ionotropic receptors?

• Nicotinic receptor

• Glutamate receptor

• P2X receptor

Nicotinic receptor superfamily

• 4 transmembrane domains

• pentameric shape = 5 subunits make up the receptor

• C terminal and N terminal in extracellular space

Glutamate receptor family

• 3 transmembrane domains

• tetrameric shape = 4 subunits make up the receptor

• C terminal in intracellular

• N terminal in extracellular

ATP P2X receptor family

• 2 transmembrane domains

• trimeric shape - 3 subunits make up the receptor

• C terminal and N terminal are in the intracellular space

• have big loop in extracellular side where ATP binds

Nicotinic - Pentameric receptors (Cys-loop type receptors)

• composed of 5 subunits

• each subunit has 4 TM domains (M1-M4)

• M2 lines the pore

• long intracellular loop between M3 and M4

• large extracellular N-terminal domain

  —> example: nicotinic acetylcholine receptor (nAChR)

neuronal nicotinic acetylcholine receptors (nAChRs) exist as combinations of what?

• a2-a10 (alpha) and B2-B4 (beta) subunits

• different subunit combinations have different affinities and locations

a4b2 (alpha 4 beta 2) nAChRs subtype

• highly expressed in the cortex and hippocampus

• high affinity for nicotine and varenicline

• plays major role in nicotine addiction

what does chronic nicotine exposure cause?

upregulation of a4b2 receptors which increases craving and dependence

what have studied shown that specific polymorphisms in subunit genes CHRNA4 (a4) and CHRNA5 (a5) are linked to?

tobacco dependence

some rare variants are linked to protection against nicotinic dependence

what is autosomal dominant nocturnal front lobe epilepsy (ADNFLE) caused by?

• mutations in M2 region of the human a4 neuronal nicotinic acetylcholine receptor (nAChR)

what do mutations in M2 region of the human a4 neuronal nicotinic acetylcholine receptor (nAChR) lead to? (mechanism)

• use dependant potentiation and a delay in the rising phase of receptor activation due to slow unblocking of closed receptors

• as a result - enhanced receptor function leads to increased nicotinic mediated transmitter release causing ADNFLE seizures

Glutamate receptors - Tetrameric assembly

• glutamate is the main neurotransmitter in the brain

• they form as dimer of dimers

• they have 4 subunits but the 2 subunits are identical to each other

• the ligand binding site has a cleft that closes when it is occupied by a ligand which forces the channel to open

what are the 3 main types of glutamate receptors?

AMPA, NMDA, Kainate

AMPA receptors

mediate fast excitatory synaptic transmission in the CNS

NMDA receptors (N-methyl-D-aspartate receptor)

involved in learning and memory

Kainate

linked to schizophrenia, depression, Huntington’s

(similar to AMPA but lesser role at synapses)

what do the variety of glutamate receptors come from?

• from different genes, alternative splicing, and RNA editing

  —> this changes the pharmacology, permeability and function

RNA splicing - flip and flop isoforms

• each AMPA subunit has two alternative splice isoforms called flip and flop - they differ by a small sequence in the extracellular ligand binding domain

• the two isoforms have different kinetic properties:

  flop = faster desensitisation + quicker channel closing

  flip = slower desensitisation + longer channel open times

• flop has reduced current response to glutamate than flip

• the brain can control how strong or long lasting AMPA signals are by choosing to express more flip or flop isoforms

If the brain wants a stronger, longer-lasting AMPA receptor signal, should it express more flip or flop isoforms?

it should express more flip isoforms, because:

• Flip isoforms have slower desensitization and longer channel open times, leading to stronger and more sustained excitatory signals

• Flop isoforms desensitize faster, causing signals to be shorter and weaker

RNA editing - GluA2 Q/R site

• the GluA2 Q/R site is located in the M2 region of the subunit inside the channel pore

• RNA editing changes:

  CAG (glutamine) → CGG (arginine) codon

• the effect of GluA2 Q/R editing: the edited GluA2 reduces Ca2+ permeability of the receptor

• mice mutant lacking enzyme (ADAR) responsible for RNA editing is more prone to seizures + early death

what is NMDA (glutamate receptors) important for an what can a dysfunction of this receptor lead to?

• important for controlling synaptic plasticity and mediating learning + memory functions

• excess stimulation of NMDA in strokes leads to neuron death

what can dysfunction of RNA modification lead to?

pathological conditions like ALS (Amyotrophic Lateral Sclerosis) and Glioblastoma

explain ALS (Amyotrophic Lateral Sclerosis) as a result of dysfunction of RNA modification

• reduced GluA2 Q/R editing in motor neurons can lead to increase in Ca2+ permeable AMPA receptors

• which can cause damage due to glutamate excitotoxicity → neuron damage

  (caused by downregulation of editing enzyme ADAR2)

explain glioblastoma as a result of dysfunction of RNA modification

• decreased ADAR2 activity = less GluA2 G/R editing

• increase in Ca2+ entry = which activates the Akt pathway promoting proliferation and tumorigenesis

• whereas editing GluA2 Q/R reverses the malignant effects = potential therapeutic target

ATP P2X receptors - trimeric assembly

adenosine triphosphate (ATP) gated ion channel

• has 3 subunits with 2 TM domains

• large extracellular domain

• needs 3 ATP molecules for the channel to open

• widely expressed

• exist as different subtypes expressed in different areas of the body → P2X 1-7 subtypes of subunits

what does the diversity and specificity of subunits allow for?

potential for drug treatments (drugs in clinic)

—> understanding the molecular mechanisms of different types of receptors of ion channels is vital for future medical advances

ionotropic receptor function

direct exchange of ions through a pore in the ion channel

Features of simple ion channels

• gated (can be non gated too)

• subunits: 4

• helices across membrane: 2

• p-loop

• function: secretion/absorption of fluids

Feature of voltage gated channels

• gated (controlled by membrane potential)

• subunits: 4

• helices: 6-24 (K+ = 6, Na+ and Ca2+ = 24)

• p-loop

• cytoplasmic anchor

• voltage sensing domains

• plugging mechanism

• function - creating action potentials in excitable cells (neurons, cardiac muscle)

Feature of TRP (transcient receptor potential) channels

• gated

• subunits: 4

• helices: 6

• p loop

• cytoplasmic anchors

• plugging mechanism

• function - hot/spicy taste

features of ligand-gated ion channel

• gated (controlled by chemical transmitters either intracellular or extracellular)

• subunits: 4

• helices: 6

• cytoplasmic anchors

• function - olfaction (cAMP), muscle (calmodulin)

P2X/Trimeric

• extracellular ligand: ATP

• example disease/physiological condition:

  P2X2 - hearing loss

  P2X4 - pain

  P2X7 - inflammation, neurodegenerative disease

Glutamate/Tetrameric

• extracellular ligand: Glutamate

• example disease/physiological condition: excess NMDA in stroke = neuron death

Cys-loop/Pentameric

• extracellular ligand: nicotinic acetylcholine, GABA, serotonin, glycine

• example disease/pathological condition: epilepsy