Voltage- and Ligand-Gated Ion Channels, University of Glasgow Presentation

Introduction to Ion Channels

Dr Graeme Sills 25 September 2024 VIA VERITAS VITA University ofGlasgow School of Cardiovascular & Metabolic Health graeme.sills@glasgow.ac.ukOverview

• lon channels as drug targets
• Structure and function of voltage-gated ion channels; focus on Na+, Ca2+ and K+ channels
• Examples of drugs that target voltage-gated ion channels
• Structure and function of ligand-gated ion channels; focus on Cys-loop receptors and glutamate receptors
• Gating mechanism of nicotinic acetylcholine receptor
• Examples of drugs that target ligand-gated ion channelsProtein targets for drug binding

A RECEPTORS Direct lon channel opening/closing Enzyme activation/inhibition Agonist/ inverse agonist Transduction mechanisms lon channel modulation DNA transcription Antagonist No effect Endogenous mediators blocked c ENZYMES Inhibitor Normal reaction inhibited False substrate Abnormal metabolite produced O Prodrug Active drug produced B ION CHANNELS Blockers Permeation blocked Modulators Increased or decreased opening probability D TRANSPORTERS Normal transport Inhibitor or Transport blocked False substrate Abnormal compound accumulated Images from: Rang & Dale's Pharmacology, 9th Edition. 2020Ion channels as drug targets

• Two major types of ion channels directly involved in drug action: - Voltage-gated ion channels (VGICs) - Ligand-gated ion channels (LGICs); considered as receptors
• Other ion channels (cell surface and intracellular) may be activated indirectly via GPCRs (not considered here)
• Passage of specific ions is determined by selectivity of channel pore
• lon flux is driven by the electrochemical gradient; direction of ion travel (influx or efflux) is determined by: - Concentration gradient (many cell types) - Electrical (or charge) gradient (excitable cells only)Electrochemical gradient

- - + + - + - + + OUTSIDE INSIDE + concentration gradient (with no membrane potential) + + + + 1 ++ + + + + +++ + ++++ +++ + + electrochemical gradient with a membrane potentialBasics of voltage-gated ion channels

• VGICs expressed in electrically excitable cells (e.g. muscle, nerve)
• Permeable to sodium (Na+), potassium (K+), calcium (Ca2+), chloride (Cl-)
• Comprise one or more a-subunit proteins that associate with ancillary subunits; modify function but not necessary for basic channel activity
• Gated (i.e. activated) by changes in membrane potential: - Responsible for action potentials (Na+, K+) - Pre-synaptic regulation of neurotransmitter release (Ca2+)
• Key targets of several drug classes; anti-hypertensives, local anaesthetics, anti-seizure drugs ....Action potential generation

Na+ Sodium channel m gate n gate Plasma membrane Potassium channel h gate K+ +50 Em (mV) 0 Resting State -50 -100 Begin depolarization V-gated K+ channels begin to open V-gated Na+ channels begin to close V-gated Na+closed V-gated K+ open K+ rushes out +30 Membrane potential (mV) Depolarization Repolarization 0 Another graded potential reaches threshold V-gated Na+ channels open Na+ rushes in V-gated K+ channels closing and the rush of K+ out slows down Stimulus causes graded potential and ligand gated cation channels open Threshold AlIV-gated K+ Closed -70 Hyperpolarization All K+ leak channels open and some V-gated K+ channels still open

Comparison of Ion Channels

Voltage-Gated vs. Ligand-Gated Ion Channels

Voltage-gated ion channels Ligand-gated ion channels Structure Monomeric (Na+ & Ca2+) Homomultimeric (K+) Heteromultimeric (3-5 independent subunits from multiple families) Ancillary subunits Common Rare Gating Depolarisation Ligand binding Endogenous ligand No Yes Selectivity High (single ion species) Low (multiple ion species) Location Excitable cells only Many cell types Drugs Mostly blockers Agonists, antagonists, allosteric modulators, etc. Activation rate ~1 millisecond ~1 millisecondStructural topology of key VGICs

Nav channels ₿2/4 Pore loop ₿1/3 II III IV Outside A n S1 S2 S3 S5 S6 + + + + + + + + + + + + Inside C a-subunit N C Na+ I IV + + $4+ +Voltage-gated ion channels

• Closed at resting membrane potential (-70mV)
• Rapidly open & close in response to changes in membrane potential
• Involved in depolarisation & repolarisation and neurotransmitter release
• Channel opening is mostly transient and rapidly inactivates
• Cycle through 3 conformational states; resting (closed), activated (open) & inactivated
• Ball & chain mechanism and changes to conformational shape of transmembrane protein Na* Channel Ca2+ Channel K* Channel Cl-Channel Na* Ca2+ CI- ions membrane potential change + Activation + + + Deactivation + Closed Open Pore of the channel + + + Recovery inactivation Closed-state Reopening Inactivation + + Inactivated + Ball and chainVoltage-gated sodium channel

Voltage-Gated Sodium Channel Drugs

• Phenytoin is a classical sodium channel blocking anti-seizure drug
• Binds preferentially to the inactivated state of the channel
• Slows conformational recycling back to resting state (does not block pore)
• Extends the refractory period between individual action potentials; reduces ability of neurons to fire at high frequency
• Local anaesthetics work in similar way but block nerve conduction completely 1 Resting (closed) 2 Activated (open) Nat Extracellular side ++ + + + + Fast channel opening + + + + + O Cytoplasmic side Inactivation gate Activation gate Slow 3 Inactivated (closed) + + + + + + SlowVoltage-gated calcium channel

Voltage-Gated Calcium Channel Drugs

• Gabapentin (GBP) and pregabalin (PGB) are newer anti-seizure drugs
• Bind to ancillary a2-81 subunit of voltage- gated calcium channel
• The a,-8, subunit associates with Ca 2.1 a-subunit to form P/Q-type channel
• GBP and PGB indirectly block the P/Q- type channel
• Involved in neurotransmitter release at synapse; glutamate? Ancillary subunits B1, B2, B3, B4 Y1 through Y8 a2-84 through a2-84 Neuronal & subunits HVA Ca,1.2 Ca,1.3 L-type Ca,1.4 a.2 Ca, 2.1 P/Q-type Ca, 2.2 N-type Ca, 2.3 R-type 8 Y LVA Ca, 3.1. Ca, 3.2 T-type B Ca, 3.3 Ca2+Ligand-gated ion channels

Ligand-Gated Ion Channel Families

• Superfamily of receptors; also known as ionotropic receptors
• Three main families of receptors; Cys-loop receptors, ionotropic glutamate receptors and ATP-gated channels
• Activation leads to a conformational change in the receptor complex that results in opening of the ion pore
• Resulting ion flux causes depolarisation (e.g. Na+ influx) or hyperpolarisation (e.g. Cl influx) of the cell membrane
• lon flux is passive and driven by the electrochemical gradient for the permeant ions Ligand Ca2+ Ligand-gated ion channel 53Gating of LGICS

Gating Mechanism of Ligand-Gated Ion Channels

• Channels opened (or gated) by binding of ligand to orthosteric site(s)
• Triggers conformational change that results in the conducting (open) state
• Modulation of gating can occur by the binding of endogenous or exogenous modulators to allosteric sites
• Mediate fast synaptic transmission, on a millisecond time scale, in the nervous system and at the somatic neuromuscular junction Gate closed olons Signaling molecule (ligand) Plasma membrane Ligand-gated ion channel receptor Gate open Cellular response Gate closedSubunit composition of LGICS

Subunit Composition of Ligand-Gated Ion Channels

• Complexes of multiple independent protein subunits assembled around a central ion pore; heteromultimers
• Heterogeneous assembly of multiple subunits; 19 GABA receptor subunits but only 5 required for functional receptor
• Subunit composition confers biophysical properties and pharmacology of the receptor complex
• Diversity of receptors; varying patterns of expression within the nervous system and other tissues
• Attractive targets for new drugs that possess subunit selectivity; i.e. discriminate between receptor isoforms a N C TM1 TM2 TM3 TM4 Single subunit b a1-6 B1-3 B GABA 71-3 Y or 8 δ, ε, θ, π BZs CI Receptor complexLGIC families

Ligand-Gated Ion Channel Families Overview

Cys-loop receptor (nicotinic ACh) superfamily NH_+ COO Pentamer Nicotinic ACh receptors 5-HT, receptors GABA, receptors Glycine receptors ZAC Glutamate receptor family P2X receptor family NH 66.56 COO NH- COO Tetramer NMDA receptors AMPA receptors Kainate receptors Trimer P2X receptorsCys-loop receptors

Cys-Loop Receptor Characteristics

• Superfamily of LGICs comprising: - Nicotinic acetylcholine receptors - GABAL receptors - 5-HT3 receptors - Glycine receptors Cys-Cys 4 4 113 3 1 1 2 3 4 4 1 3 1 3 4 cr
• Pentameric structure; usually 2 alpha-subunits plus 3 others
• Each subunit contains large extracellular N-terminal domain (with Cys-loop)
• 4 membrane-spanning alpha helices (M1-M4); pore is formed by the M2 helices
• Endogenous ligands bind at interface between subunits in the extracellular domainNicotinic acetylcholine (ACh) receptor

Nicotinic Acetylcholine Receptor Function

• First ligand-gated ion channel to be purified & cloned
• Expressed at neuromuscular junction (NMJ), autonomic ganglia and in the CNS
• Two binding sites for ACh; at interface between a- subunits and their neighbours
• Both sites must bind acetylcholine molecules for receptor activation
• Activation results in fast synaptic transmission A ₿ 8 ACh ACh 6 nm Exterior Membrane 3 nm Cytosol 2 nm - cf-Helices forming gate 8 ACh Pore ~0.7 nm diameter 0 ACh Y 0 CH3 H2 C +N H3C O C H2 ·CH3 CH3 AcetylcholinenAChR - a non-selective cation channel

Nicotinic Acetylcholine Receptor as a Non-Selective Cation Channel

Polarized postsynaptic membrane (~ - 75 mV) Depolarized postsynaptic membrane (~ 0 mV) Na+ × + + + + High [K+] Low [Na+] V K+ Acetylcholine Direction of action potential

• Resting membrane potential of post-synaptic cell is ~- 70mV
• High Na+ concentration outside cell, high K+ concentration inside cell
• Pore of the nicotinic receptor is equally permeable to all monovalent cations (e.g. Na+, K+, Li+); non-selective
• Opening of pore allows ions to flow down concentration gradient; Na+ in, K+ outGating of nicotinic ACh receptor

Gating Mechanism of Nicotinic Acetylcholine Receptor

• Gating is highly dependent on presence of specific amino acid residues in pore
• Glutamate and threonine residues that line the pore attract cations and repel anions
• Each subunit has a leucine residue in M2 helix; protrude into pore to form the gate
• In the resting state, leucine residues block the pore - gate closed channel + + + + + pore 1 gateGating of nicotinic ACh receptor

Nicotinic Acetylcholine Receptor Activation

• Two ACh molecules bind to extracellular domain of a-subunits
• Conformational change - twisting of a- subunits, M2 helices swivel out of the way
• Gate opens - channel activated
• Cations pass down concentration gradient into cell (25,000 Na+ ions per millisecond)
• K+ ions move in opposite direction; 3 Na+ ions enter for every 2 K+ ions leaving
• Net increase in intracellular positive charge; leads to depolarisation of membrane channel ACh ACh + + + + + pore U + + + + +