pcol 6.25 repeat Receptor Pharmacology – Ligand-Gated Ion Channels & GPCRs

Historical Foundations

• 1905 John Langley: observed that some substances/hormones produce effects without crossing the cell membrane → postulated a “receptive substance” on the surface.
• 1909 Paul Ehrlich: coined the term receptor.

Working Definition of a Receptor

• Macromolecular complex (protein ± accessory sub-units).
• Binds endogenous ligand (neurotransmitter, hormone) or drug (agonist/antagonist).
• Ligand binding ➜ conformational change ➜ activation/inactivation of downstream signalling pathway ➜ physiological response.

Major Plasma-Membrane Drug-Target Receptors Covered

1. Ligand-gated ion channels (LGICs, “ionotropic”).
2. G-protein-coupled receptors (GPCRs, “metabotropic”).
3. (Preview only) Enzyme-linked receptors, cGMP-linked receptors, nuclear hormone receptors, transcription factors.

Ligand-Gated Ion Channels (LGICs)

• Alternative names: channel-linked receptors, ionotropic receptors.
• Coupled directly to an ion pore; channel opens/closes when an agonist (ligand) binds.
• Kinetics: “fast” receptors – responses in milliseconds (≪ GPCRs).
• Pore diameter larger than voltage-gated channels → lower ion selectivity; direction of ion flow governed by electro-chemical gradients.
• Typical roles: neurotransmission, cardiac conduction, skeletal-muscle contraction — all processes needing split-second timing.

Structural Blueprint

• Oligomeric pentamer: 5 sub-units arranged around a central pore.
  • Mandatory: 2 α sub-units (contain ligand-binding sites).
  • Accessory: β, γ, δ, ε, θ, etc. → sub-unit stoichiometry varies by tissue & developmental stage.
• Each sub-unit spans the membrane 4 times (M1–M4 helices).
  • 5\ \text{sub-units}\ \times\ 4\ \text{TM domains}=20 total TM helices per receptor.
• The M2 helix of every sub-unit lines the pore → forms the activation “gate.”
• Positive cooperativity: binding of first agonist molecule to α1 increases affinity of α2; both sites must be occupied to open the channel ⇒ need 2 agonist molecules.
• Conformational change mechanism:
  • Resting state: kinked M2 helices & bulky side chains project into pore → occlusion.
  • Agonist binding → helices “splay” outward → pore widens → ion flux.

Ion Flux Logic

• Na⁺: high EC [Na⁺] & negative membrane potential → massive inward drive.
• K⁺: concentration gradient outward, electrical gradient inward → only modest efflux during LGIC opening (crowded doorway analogy).
• Cl⁻: moves inward if channel is Cl⁻-selective & membrane potential is above ECl, hyperpolarising the cell.

Representative LGIC Families

Excitatory Channels

• Nicotinic acetylcholine receptor (nAChR)
  • Ligand: acetylcholine (ACh); also sensitive to nicotine.
  • Tissue-specific stoichiometries:
  • Neuromuscular junction (NMJ): \alpha1\alpha1\beta1\delta\gamma (newborn) → \alpha1\alpha1\beta1\delta\varepsilon (adult).
  • Autonomic ganglia: two α (α2–α10 variants) + three β (β2–β4) sub-units.
  • Pharmacological significance: NMJ blockers can be tailored to this sub-unit pattern to avoid autonomic side-effects.
• Ionotropic glutamate receptors – e.g. NMDA, AMPA (mentioned briefly): glutamate = ligand; mediate excitatory CNS transmission.

Inhibitory Channels

• GABA_A receptor
  • Typical sub-unit set: \alpha1\alpha1\beta2\beta2\gamma_2 (multiple isoforms exist).
  • Ligand: GABA (γ-aminobutyric acid).
  • Opens Cl⁻ pore → Cl⁻ influx → hyperpolarisation (membrane potential moves further from threshold) → decreased action-potential frequency.
  • Allosteric modulators:
  • β sub-unit sites bind barbiturates.
  • γ sub-unit site binds benzodiazepines.
  • Result: left-shift of GABA dose–response curve (higher potency) — molecular “tail-wind” analogy.
• Glycine receptor – ligand: glycine; Cl⁻ influx; spinal cord & brainstem inhibition.

Rapid-Fire Self-Check (from lecture polls)

• How many TM domains per LGIC? – 20.
• How many agonist molecules required to activate a pentameric LGIC? – 2.

G-Protein-Coupled Receptors (GPCRs)

• Also called metabotropic receptors.
• ~800 genes in the human genome ⇒ largest drug-target family.
• Produce effects in seconds → minutes (“relatively fast,” slower than LGICs).
• Broad physiological scope: sensory perception, autonomic control, hormone & peptide signalling, smooth-muscle tone, secretion, cardiac modulation, CNS circuits.

Signature Architecture

• Single polypeptide with 7 trans-membrane (7TM) α-helices.
  • Extracellular N-terminus; intracellular C-terminus.
  • Ligand-binding pocket formed primarily by TM 3–6 residues.
  • Third intracellular loop (IL3) = G-protein coupling domain.
• Ligand examples (examination list):
  • Muscarinic (M1–M5) ACh receptors – activated by muscarine.
  • Biogenic-amine receptors: adrenergic (α, β), dopaminergic (D1–D5), serotonergic (5-HT), histaminergic.
  • GABA_B, opioid (μ, κ, δ), peptide-hormone receptors, etc.

Heterotrimeric G-Protein Cycle

1. Basal state: receptor empty; G-protein (αβγ) anchored to membrane; GDP bound to α.
2. Agonist binds receptor ➜ conformational change ➜ receptor acts as GEF (guanine-nucleotide exchange factor): GDP released, GTP binds α.
3. Dissociation:
   • \alpha{-}GTP separates from \beta\gamma dimer.
4. Effector modulation:
   • \alpha{-}GTP stimulates or inhibits enzymes (AC, PLC) or channels.
   • \beta\gamma can also regulate K⁺/Ca²⁺ channels & PI3-kinase.
5. Intrinsic GTPase of α hydrolyses GTP → GDP (accelerated by RGS proteins – Regulators of G-protein Signalling).
6. α-GDP reassociates with βγ → ready for next cycle (continues while ligand remains bound).

Canonical Effector Pathways & Second Messengers

Additional βγ effects: open GIRK (G-protein-activated inward-rectifier K⁺) channels, inhibit N-type Ca²⁺ channels, activate PI3-kinase.

Why Phosphorylation Works

• Phosphate group carries 3 negative charges → drastic electrostatic & conformational change in substrate protein.
• Reversible toggle:
  • Kinases add PO_4^{3-} (ON switch).
  • Phosphatases remove it (OFF switch).

First vs. Second Messenger Concept

• 1st messenger = extracellular ligand (does not cross membrane).
• 2nd messenger = small intracellular molecule generated enzymatically (e.g.
  cAMP, IP_3, DAG, Ca^{2+}) that amplifies & distributes the signal.

Determinants of Signalling Specificity (Pharmacological Relevance)

• Ligand selectivity (e.g. epinephrine β ≫ α).
• Tissue-selective receptor expression & sub-unit composition.
• Differential G-protein coupling (Gs vs Gi vs Gq).
• Distinct regulatory proteins (GRKs, arrestins, RGS).
• Receptor dimerisation (homo- or hetero-) alters ligand profile & signalling bias.
• Exploited in drug design to maximise therapeutic action & minimise off-target effects (e.g. NMJ blockers sparing autonomic ganglia).

Key Numerical & Conceptual Take-Aways

• LGIC pentamer: 5 sub-units, 4 TM helices each → 20 helices total.
• Need 2 agonist molecules bound (both α sites) for pore opening.
• LGIC responses: milliseconds; GPCR responses: seconds-minutes.
• Human genome encodes ≈ 800 GPCRs.
• GPCR α-sub-units: Gs ↑cAMP; Gi ↓cAMP; G_q ↑DAG/IP₃ (↑Ca²⁺).
• Phosphorylation = reversible, charge-based molecular switch controlling protein function.

Conceptual Flow Summary

1. Extracellular ligand binds receptor (LGIC or GPCR).
2. Receptor undergoes conformational change.
   3a. LGIC: pore opens instantly → ion flux → rapid depolarisation/hyperpolarisation.
   3b. GPCR: activates heterotrimeric G-protein → enzymes/channels → second messengers → protein phosphorylation.
3. Physiological effect (muscle contraction, neurotransmission, heart rate change, secretion, etc.).
4. Termination mechanisms: ligand unbinding, GTP hydrolysis, RGS proteins, phosphatases, receptor desensitisation & internalisation.

Mastery of these molecular mechanisms underpins rational drug therapy, prediction of side-effects, and future design of receptor-selective agents.