Lecture 13-15: Ligand-Gated Ion Channels & Transporters

Excitatory Ligand-Gated Ion Channels

Na⁺ / Ca²⁺

• Glutamate (NMDA, AMPA, Kainate)

• Nicotinic acetylcholine

• Serotonin 5-HT

• Purinergic P2X

• Vanilloid (capsaicin)

• Transient receptor potential (TRP)

Inhibitory Ligand-Gated Ion Channels

Cl^-

• GABA_A (also HCO₃^-)

• GABA_C

• Glycine

Cys-Loop Superfamily

• Does not include Glu R

• 18-24% overall sequence homology (greatest in TM regions)

• Disulphide bonds stabilize tertiary structure

• Large N-terminus (>200aa) with potential glycosylation sites

• 4 TM domains, large IC loop between TM3/4 with potential phosphorylation sites

• 5 subunits form pentamer around central M2 region pore

Nicotinic Cholinergic Receptor

Endogenous ligand: Acetylcholine — also binds nicotine with high affinity

Binding promotes Na⁺ influx down electrochemical gradient

Subunit composition determines channel conductance (what current moves through), mean open time, pharmacological profile (which drugs bind and their effects)

B subunit important with dissociation rates of Ag/Ant, rate of receptor opening

Nicotinic Receptor Activation

• TM gate prevents ion flow at rest

• 2 Ach bind (one to each a subunit), increase probability of open conformation

• Adult muscle nAchR at NMJ: 2 a subunits, B, y/e, d subunits

• Neuronal nAchR have no y, d, e

Torpedo Electric Organ

Derived from modified nerve/muscle tissue, high nAchr expression

• Isolated hetero-oligomeric muscle nAchR pentamer (~250kDa), localized to endplate

  • Like localization in NMJ motor endplate

• Channels open within 0.3ms of Ach release

• Non-selective cation channel (<6.5Å)

  • Na⁺ conductance: 10⁷ ions/channel/sec

  • Ca²⁺ conductance increases with a3/5, largest with a7

Ach Binding Sites

• a-y and a-d interface on peripheral receptor

  • NMJ: Greatest Na⁺ conductance

• 2 sites on a-B interface of neuronal receptors

  • CNS/ganglia: Relatively equal Na⁺ / Ca²⁺ conductance

• 5 identical sites on homomeric a7, a8, a9 receptors

  • CNS, some ganglia: 10:1 Ca²⁺ / Na⁺ conductance

• High (~10nM) and low (~1um) affinity sites

nAchR Closed State

Low probability of ion movement, few channels open

nAchR Activated State

Initiated by Ach binding both high and low affinity sites for a short period of time, increased open probability (us-ms after binding)

nAchR Closed and Desensitized State

Phosphorylation/conformational change after prolonged Ach exposure and long-term binding of both sites OR when low affinity site not occupied

Neuronal nAchR

Only a2-9, B2-4

Receptor activation → Ca²⁺ increase → cell signalling → gene expression → exocytosis, memory, addiction, survival, development

Physiological regulation: Cognition, learning & memory arousal, cerebral blood flow, metabolism, skeletal muscle contraction

Neuronal nAchR Channelopathies

• Autosomal dominant frontal lobe epilepsy (a4, a7)

• Schizophrenia (a7)

• Alzheimer’s (a4, a7)

• Parkinson’s

• Tourette’s

• Anxiety

• Depression

• Myasthenia Gravis

• Nicotine use dependence (a4B2)

Nicotinic Agonists

Ach, nicotine (Varenicline used to help with smoking cessation, pAg @ a4B2), succinylcholine

Nicotinic Antagonists

d-Turbocurarine (curare) @ NM, a-bungarotoxin (irreversible)P

Positive Allosteric Modulators (PAM)

Physostigmine/Neostigmine (organophosphates), a7 PAM

Negative Allosteric Modulators (NAM)

Chlorpromazine, local anaesthetics, steroids, ethanol, barbituates

Phosphorylation

PKA, PKC, TK

Potential of Subunit-Specific PAM

a7 PAM or a4B2 PAM

Enhance endogenous Ach, DMXB-A

Analgesia: Treatment of acute, chronic, neuropathic pain

Pro-cognitive effects: Increased attention, reduced cognitive deficits

Neuroprotection: Increased resistance to cerebral ischemia

Anti-inflammatory: Inhibition of TNFa

nAchR and NMJ

Concentrated regions of nAchR on folded postjunctional membrane of skeletal muscle — invaginations increase surface area for expression

90% of protein at postjunctional fold is nAchR

Action potential triggers Ach release from vesicles, interaction with nAchR

Acetylcholinesterase stimulates Ach metabolism into acetate and choline, released into the blood or taken back up by neuron

Neuromuscular Blocking Agents (NMBA)

Block cholinergic transmission between motor nerve endings and nAchR on neuromuscular endplate of skeletal muscle

Non-depolarizing block: Competitive transmission blockade, no nAchR activation

Depolarizing block: Initial activation of nAchR, then blockade (desensitization) — agonists or acetylcholinesterase inhibitors

Bungarotoxin

Identified in venom of Southeast Asian krait snake (Bungarus multicinctus) — snake nAchR mutated for decreased affinity for toxin

a-bungarotoxin: Irreversibly binds and blocks nAchR → neuromuscular blockade, skeletal muscle paralysis

B-bungarotoxin: Prevents release of Ach from motor nerve endings → death by respiratory paralysis

Curare/D-turbocurarine

Non-depolarizing block

From extracts of root bark, vines indigenous to South America (used in hunting)

D-turbocurarine = active ingredient, first used as muscle relaxant in anesthesia in 1940s

• Competes with Ach for binding sites on nAch receptor (less effective at high Ach levels)

• Acts at areas outside NMJ, may cause respiratory depression and blood pressure decrease from histamine release (immune response)

• Onset of action and duration long relative to desired clinical use

Succinylcholine

Agonist, 2 molecules of Ach coupled together

1. AchR binding causes activation, muscle cell end-plate membrane depolarization

   • Transient twitching of muscles/fasciculations

   • Cannot be broken down by acetylcholinesterase, constant receptor stimulation

2. Membrane repolarizes, but continued binding renders R incapable of further impulses

   • Endplate remains depolarized as muscle relaxes, unresponsive to stimulation

   • Flaccid paralysis

Curare vs. Succinylcholine — Muscle Fibre Twitch

Curare:

• Single twitch more sensitive than tetanic stimulation

• Interference in skeletal muscle contraction when 75-80% nAchR occupied

• Complete block at 90-95% occupancy

• Reversed by neostigmine: Indirect PAM inhibits Ach esterase, increases Ach at cleft to outcompete curare

Succinylcholine

• Tetanus more sensitive to succinylcholine

• Ach esterase inhibition by neostigmine enhances response: More Ach in cleft → further desensitization

Applications of nAchR Modulation

• Subtype selective: a7B2 activity in neuroprotection

• pAg (Cytisine, varenicline): Smoking cessation, act on mesolimbic & reward pathways

• Positive modulators: Cognition, neurodegeneration, schizophrenia (a7 selective)

• Pain: Epibatidine (a4B2 Ag)

• Muscle relaxation: Depolarizing block vs. non-depolarizing/competitive antagonists

• Ganglionic blockade

  • Myasthenia Gravis

GABA_A Allosteric Modulation Sites

Neurosteroids, barbiturates, alcohol, anesthetics, benzodiazepines

BZD Agonism

Partial or full

Eg. Diazepam = PAM

BZD Inverse Agonism

Eg. Ro-154513 = NAM

BZD Antagonism

Eg. Flumazeinil (treatment for OD/coma, no effect on its own)

BZD Anxiolysis and Sedative-Hypnotic Agonism

Increase frequency of channel opening, affinity/potency of GABA for receptor, modulate gating, alter desensitization → increased Cl^- conductance

Shift GABA dose-response curve left

Beta-Carbolines

Reduce influx of Cl^- below baseline state, increasing excitability

Accompanied clinically by anxiety

Eg. Ro-154513

Receptor Subtype Selective Pharmacology

Properties dependent on subunit composition (a1B2y2 most common in cortex(

BZD modulation dependent on subunit composition and BZD used

Diazepam Binding

Insensitive to a4/a6 containing receptors

High affinity binding pocket in a1 His101

Site-directed mutagenesis/point mutation at His101 blocks sedative effects

Zolpidem/Ambien

NonBZD structure acting at BZD site

a1-containing GABA (globally in CNS): Sedation

a2 and a3 GABA_A

Anxiolytic

Inverse agonist at a3: Anxiogenic

a2

Schizophrenia

a5

Learning and memory

NAM: Basmisanil

Anxiety

d and B3

Anaesthetics, sleep

Issues with Benzodiazepines

• Misuse due to tolerance/dependence and action in “reward” system (VTA, NAcc)

• Tolerance to sedatie effects faster than to anticonvulsant/anxiolytic actions

  • Receptor expression, turnover, changes in subunit expression, post-translational changes → need more dose or other agonists (eg. alcohol)

Benzodiazepine Development

244 patent submissions since 1998: Brain injury, eating disorders, gut motility

Highly selective GABA_A R modulators: Fast-acting antidepressants, anticonvulsants for pediatric epilepsy, treatments for autism, cognitive disorders

Neurosteroid Modulation

2019: Brexanolone (allopregnanolone) for postpartum depression (PPD)

60 hours IV infusion improves PPD, replenishing allopregnanolone levels and normal GABAergic function, which may be altered after childbirth

Neurosteroids have higher affinity for a2/a5, a4, d subtypes involved with tonic inhibition → directly activate GABA_A R, modulate frequency, open time, and prolong delay

a2 mice with knock in NS insensitivity have increased anxiety, depressive behaviours

Glutamate Receptor Structure

Glu is endogenous ligand for all iGluR

Only 3TM regions — 2nd TM region is hairpin loop within membrane (discovered by Hollmann et al. in 1994 using novel N-glycosylation sites

Predominantly heteromeric and homomeric receptors

Subunits undergo post-translational modifications

iGluR ion Selectivity

Ion selectivity: Na⁺, K⁺, Ca²⁺

NMDAR most permeable to Ca²⁺ (Mg²⁺ block)

AMPAR and kainate (KA) receptor Ca²⁺ permeability proportional to RNA editing and alternative splicing

iGluR Ca²⁺ Influx Clinical Targets

LTP, LTD, excitotoxicity, low [ ] trophic effects, high [ ] toxic effects, activation of IC enzymes, osmotic swelling

Bone deposition, wound healing, insulin secretion, BBB integrity, myelination

Diseases/Disorders Related to Glu Activation via NMDAR/AMPAR & Glia

Huntington’s, Parkinson’s, Alzheimer’s, psychosis, schizophrenia, ALS, epilepsy, stroke

Neuropathic pain, dementia, melanoma, neuroprotection

Substance use disorder (maladaptive learning)

Cancer

GI disorders

Pharmacological Agents Acting via iGluR

PCP/ketamine: Hallucinogen, dissociative anesthetic “special K”

New NMDAR antagonists (blunt Ca²⁺ entry for neuroprotection): Schizophrenia, Alzheimer’s disease (memantine), depression

AMPAR antagonists: Epilepsy, ischemic stroke

AMPAR positive modulators: Cognitive enhancement

Uptake Transporters (Solute-Linked Carrier/SLC Superfamily)

• Organic anion transporters (OATs)

• Organic anion transporting polypeptides (OATPs)

• Organic cation transporters (OCTs)

• Organic cation transporter novel type (OCTNs)

Efflux Transporters (ABC Superfamily)

• Multidrug resistance proteins (MDRs)

• Multidrug resistance-associated proteins (MRPs)

  • Breast cancer resistant protein (BCRP)

Renal Filtration

25000 mmol Na⁺ daily

Nephron: Functional unit of kidney

Glomerulus: Electrolyte, water, waste product, small hormone filtration from blood

Renal Na⁺ Transport

Proximal convoluted tubule: Reabsorption of glucose, amino acids, urea, NaHCO₃, 60% of filtered water, 40% of filtered NaCl, much more → 65% Na⁺ reabsorbed

Thin descending Loop of Henle: Salty interstitium of medulla draws water out of tubule

Thick ascending Loop of Henle: Reabsorption of NaCl via NKCC2, impermeable to H₂O, dilution of tubular fluid → 25% Na⁺ reabsorbed

Distal convoluted tubule: Reabsorption of NaCl via NCC, impermeable to H₂O, dilution of tubular fluid → 5% Na⁺ reabsorbed

Collecting tubule: Reabsorption of small amounts of NaCl, volume adjustments, secretion of K⁺ → 2% Na⁺ reabsorbed

SLC12

Electroneutral cation-chloride transporter family

9 transporters, all involved in cotransport of Cl^- and an electroneutral amount of Na⁺/K⁺

NKCC2

• SLC12A1, kidney-specific

• 12 TM domains, large EC loop between TM7-8

• 2 glycosylation sites

• 2 large IC domains

• Likely functions as a dimer

• 3 isoforms (A, B, F) differ in kinetics and TAL locations

• Modulated via cAMP levels from various GPCRs, vasopression, glucagon, PTH

NKCC2 Na⁺ Reabsorption at TAL

• Basolateral Na⁺ / K⁺ ATPase pumps Na⁺ out, K⁺ in

• NKCC2 transports Na⁺ down its concentration gradient @ apical/luminal membrane

• Drives co-transport of K⁺, Cl^-

• Buildup of IC K⁺ leads to K⁺ recycling — secretion back into lumen via leak channels

• +ve lumen drives transport and paracellular uptake of Mg²⁺, Ca²⁺

Loop Diuretics

Furosemide, torasemide

Indirectly inhibit NKCC2 @ TAL by blocking NaCl reabsorption at H₂O impermeable segments and impairing H₂O clearance

Decrease K⁺ / H⁺ secretion, indirectly inhibit Ca²⁺ and Mg²⁺ reabsorption and increase excretion

Indications: Heart failure, hypertension, edema, liver cirrhosis

Loop Diuretics PK

Rapid, incomplete GI absorption

PO onset within 30 min, 4-6 hour duration

Excreted unchanged in urine @ proximal renal tubule (target site)

Loop Diuretics Adverse Effects

• Intravascular volume depletion

• If patient with reduced GFR, will decrease further with BP/blood volume

• Hypokalemia: Metabolic alkalosis

• Hyperuricemia: Diuretics moved to tubule by transporter that moves urea into urine → uric acid in blood

• Acute rapid infusion may lead to transient deafness (NKCC1 in inner ear)

NCC

• SLC12A3, kidney-specific

• Large (approx. 1000aa)

• 12 TM domains, glycosylated EC loop (TM7-8)

• Potential dimeric structure

• Polymorphisms associated with hypertension, issues with NaCl and BP control

NCC Na⁺ Reabsorption at DCT

Na-Cl cotransport at apical membrane

Activity not associated with K⁺ recycling

No passive reabsorption of Ca²⁺ or Mg²⁺

Activity inversely associated with Ca²⁺ reabsorption (basolateral transporter)

Thiazide Diuretics

Hydrochlorothiazide: High potency, 12.5-50mg/day

Moderate efficiency, block NCC @ DCT

Impair urinary dilution

Increase K⁺ secretion via increased Na⁺ / H₂O delivery to collecting duct

Decreased Ca²⁺ excretion, stimulation of Ca²⁺ reabsorption in DCT (opposite to loop diuretics, ideal for elderly — osteoporosis)

Indications: First line treatment of hypertension, edema, heart failure

Thiazide Diuretics PK

Onset of actions ~1-3 weeks

12-24 hour duration

Excreted unchanged in urine

Thiazide Diuretics Adverse Effects

Hyponatremia, hypokalemia, hypourioemia, hypercalcemia

Diuretics and K⁺ Wasting in Collecting Tubule

Principal cells have Na⁺ and K⁺ ion channels, not co-transporters → Na⁺ reabsorption must be electrically balanced by K⁺ efflux

Diuretics increase Na⁺ delivery to collecting tubule → K⁺ wasting and hypokalemia → muscular, cardiovascular, respiratory toxicity

Sodium-Glucose Co-Transporter

SGLT1-6

SGLT2 (SLC5A1) symporter on luminal surface of PCT: 90% of glucose reabsorbed in PCT

• 670 residues in 14 TM helices

• Electrogenic, move glucose against concentration gradient (1 Na⁺ out / 1 glucose in)

• Independent of insulin

• High capacity, low infinity

SGLT2 Inhibitor: Empagliflozin

• Decreases glucose reabsorption 30-50%

• Used to help manage type 2 diabetes

• Once daily dosing (t_1/2 ~ 13 hours), monotherapy or combination

• Decrease AP duration through VG_Na (late Na⁺ current, same site as lidocaine), K_ir (inward rectifying K⁺ current)

SGLT Inhibitor Adverse Effects

Euglycemic ketoacidosis: Metabolic acidosis, decreased blood pH within normal glucose levels

Neurotransmission

Endogenous NTs transmit messages from neuron to target cell across EC synaptic cleft

NTs synthesized in presnyaptic cell, stored in vesicles for release → bind receptors on postsynaptic cell to continue transmission

Presynaptic NT transporters

Take up NTs from synaptic cleft to help terminate transmission

• Enzymatic degradation (Ach)

• SLC transporter family (Glu)

• SLC6 transporter family

SLC6 family

Sodium and chloride dependent NT transporters

• 12 TM domains

• Na⁺ influx down concentration gradient powers NT influx

• Cl^- cotransport sometimes required

• K⁺ efflux required in addition to Na⁺ influx for SERT

16 transporters, targets for over 30 drug compounds involved in treating MDD, anxiety disorders, ADHD, obesity

SLC6 KO mice

Increased EC DA/NE levels, prolonged EC lifetime

Decreased NT storage, replaced by release into synaptic cleft

Increased presynaptic NT synthesis unable to compensate for decreased storage

Norepinephrine Transporter

NET/NAT, SLC6A2

Dopamine Transporter

DAT, SLC63

Na⁺ and dopamine symporter, based on gradient from Na⁺/K⁺ ATPase

Regulated by PKA/PKC phosphorylation, internalization

Serotonin Transporter

SERT, SLC6A4

Binds and transports Na⁺, Cl^-, 5HT into presynaptic cells (and K⁺ out), based on gradient from Na⁺/K⁺ ATPase

Dopamine

Associated with movement, motivation, reward, mood, learning

Pathologies: Schizophrenia, Parkinson’s, ADHD, OCD, drug misuse

Cocaine

Inhibits DAT, SERT, NET/NAT fairly equally

• DAT inhibition: Rewarding effets

• SERT inhibition: Appetite suppression, sleeplessness, hyperactivity

• NET/DAT inhibition + SNS: Tachycardia, hypertension

Amphetamines

Amphetamine, methamphetamine promote reversal of DAT/SERT/NET direction, impairing transport into presynaptic vesicles

• Competitively inhibit dopamine uptake by utilizing DAT

• DA levels: Euphoria, abnormal movement, psychosis

• Serotonin levels: Hallucinations, hyperthermia

Ecstasy (MDMA) similar, more selective towards SERT

Serotonin

Associated with mood, anxiety, sleep, appetite, temperature, pain, eating behaviour, sexual behaviour, movement, gastrointestinal motility

Pathology: Depression, panic/OCD/PTSD/ED, schizophrenia, sleep disorders, serotonin syndrome

SSRIs

Selective serotonin reuptake inhibitors

• Fluoxetine (Prozac, 2nd gen), sertraline (Zoloft)

• Clinically used for treatment of depression, anxiety, OCD, eating disorders

• Improved safety profile vs. TCA and MAOI

  • Less anticholinergic, cardiac effects

• Delayed efficacy (several weeks) for improved mood and well-being

• Unwanted effects: Anxiety, headache, somnolescence, nausea, sexual dysfunction, weight gain, suicidal ideation

SLC6 Transporter Inhibitor Selectivity

• TCA: SERT, NET/NAT

• SSRI: SERT

• Bupropion: DAT, NET/NAT

• SNRI: NET

• Cocaine: SERT, DAT, NET/NAT