Drug Interactions with Receptors and Ion Channels (Receptors) Flashcards
What is the process of isolating nAChRs?
- nAChRs are solubilised.
- Preparation containing nAChRs added to column containing gel beads with immobilised gallamine (nAChR antagonist) on surface.
- nAChRs bind to beads,
- Excess of soluble gallamine added to column to outcompete immobilised gallamine for binding to nAChRs.
What is the structure of the nAChR?
- Pentameric protein consisting of 5 subunits (2 x α, β, γ, δ).
- Each subunit has a highly conserved structure (~50% homology), each containing 4 transmembrane α-helical domains (M1-4).
- M2 domain lines the pore of the channel and kinked inwards so that only conformational change when ACh bound opens channel.
- 2 ACh binding sites, located at the α-γ and α-δ interfaces.
What are the different families of LGICs?
- Cys-loop receptor family: Pentameric receptors. E.g. nAChR, GABAA receptor.
- Tetrameric receptor family: Tetrameric receptor with 3 full PM spanning domains and 1 re-entrant domain (M2). E.g. iGluR (glutamate receptor).
- Trimeric receptor family: Each subunit has 2 domains fully spanning PM. There are 2 variants:
- Homo-trimeric: Identical subunits. E.g. P2X1, P2X7.
- Hetero-trimeric: Different subunits. E.g. P2X2, P2X3.
How is ionic selectivity of LGICs determined?
- Net charges on receptors.
- nAChR has net charge of -66mV and so is selective for +ve Na+ and K+ ions.
- GABAA has net charge of +34mV and so is selective for -ve Cl- ions.
What is the structure of GPCRs?
- They are 40-50 kDa in size.
- Made up of 7 transmembrane α-helical domains in a circular formation, forming a central pore.
- Central pore involved in ligand binding.
- 3rd intracellular loop and C-teminus involved in interactions with G-protein.
- There’s putative phosphorylation sites on the C-terminus involved with desensitisation of the receptor.
What is the structure of G-proteins?
- α-subunit: 39/42 kDa and contains GTP binding site.
- β & γ subunit: 35 kDa and 8 kDa respectively. These are usually found together in dimer.
- All G-protein subunits are anchored to the PM.
What are the types and functions of α subunits?
- αs: Stimulates adenylyl cyclase
- α1: Inhibits adenylyl cyclase
- αq/11: Stimulates PLC (activates IP3 pathway)
What are the functions of βγ subunit?
- Inibits α subunit activity by sequestering free α subunits and deactivating them.
- Direct interactions with ion channels and adenylyl cyclase.
What is the G-protein cycle?
- Ligand binds to the GPCR, inducing a conformational change that reveals the intracellular G-protein binding site.
- Binding of the G-protein to the GPCR catalyses the exchange of GDP for GTP on the Gα subunit.
- When GTP binds to Gα, the γ phosphate interacts with switch regions, causing dissociation of Gβγ subunit from Gα and revealing the adenylyl cyclase binding domain on Gα.
- Gα also has intrinsic GTPase activity that slowly hydrolyses the bound GTP to GDP. When this happens, the switch region is able to interact with the βγ subunit again to reform the inactivated GDP-bound G-protein.
- G-proteins rebinds to GPCR and cycle repeats.
What are molecules that disrupt G-protein function?
- Cholera toxin: Causes ADP-ribosylation of αs subunit and inhibits intrinsic GTP activity, causing tonic activation of αs and adenylyl cyclase.
- Pertussis toxin: Causes ADP-ribosylation of αi subunit, preventing activation.
- AlF4- Mimics γ-phosphate on GTP, causing persistent activation of G-proteins.
What are the functions of the breakdown products of PIP2?
- IP3: Binds to intracellular IP3 receptors and opens ER Ca2+ channels, inducing Ca2+ release into cytosol.
- DAG: Activates PKC which phosphorylates a number of intracellular proteins to produce number of different effects. E.g. Phosphorylates NHE3 in renal tubular epithelium and increases acid secretion).
What is the function of lithium?
Inhibitor of inositol-1 phosphotase, which normally converts IP to inositol, and blocks inositol recycling in brain. Due to BBB, brain cannot use inositol in blood and so effects of IP3 pathway inhibited.
What are the mechanisms of β-adrenoteceptor desensitisation?
- Uncoupling: Even when extracellular agonist is bound, receptor is unable to activate G-proteins (usually due to phosphorylation of intracellular domains). This is key mechanism, with all other mechanisms only occuring following this one.
- Sequestration: Receptors are internalised into endosomes by endocytosis, or shed from cell by exocytosis.
- Down-regulation: Reduction in total number of receptors in PM as a result of increased rate of lysosome-mediated breakdown and decreased rate of synthesis.
What is homologous desensitisation?
Continuous stimulation of a certain type of receptor results in down-regulation of that type of receptor only.
What is the mechanism of homologous desensitisation?
- Gβγ stimulates βARK (β-adrenoreceptor kinase), which phosphorylates the receptor at a putative phosphorylation site on the C-terminus.
- This phosphorylation predisposes the GPCR to β-arrestin binding (on agonist occupied receptors only).
- β-arrestin prevents the binding of the Gα to the GPCR, thus uncoupling the G-protein from the receptor.
What is significant about the kinetics of homologous desensitisation?
β-arrestin only binds to agonist-bound receptors, and so the amount of desensitiation is proportional to the number of occupied receptors. This means that high [agonist] are needed to produce significant desensitisation.
