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Competitive antagonist for nAChR, used experimentaly becaise of its high affinity
alpha-bungarotoxin
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Used to trap NAChR on gel beads
Gallamine
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3 NAChR agonist with different efficacies
- ACh: 1ms open time
- Carbachol: 0.5ms
- Suberyldicholine: 1.7ms
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3 types of ligand gated ion channels, with examples
- pentameric: AChR, GABAA, Glycine, 5-HT
- tetrameric: AMPA, NMDA
- trimeric: P2X for ATP
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Which parts of GPCRs interact with G proteins
- 3rd intracellular loop (PKA)
- C-terminal tail (PKA, betaARK)
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3 types of G proteins
- Gs: stimulates AC -> cAMP (beta)
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Cholera toxin effect
- Ribosylates ADP in alpha-s and inhibits GTPase activity
- -> sustained activation of AC
- -> high cAMP
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Pertussis toxin
- Ribosylates ADP in alpha-i and prevents its activation
- -> sustained activation of AC (no inhibition
- -> high cAMP
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AIF4-
- Mimics gamma-phosphate of GTP
- -> sustained activation of G proteins (and AC)
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Agonists for phosphoinositide pathway
- alpha 1 adrenoceptor
- muscarinic AChR
- substance P
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Lithium importance in phosphoinositide pathway
used for?
- Uncompetitively inhibits phosphatase that converts IP3 to inositol
- -> blocks recycling of inositol, important in the brain as inositol cannot cross BBB
- Used in treatment of schizophrenia
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How to label calcium?
Fura-2: fluorescent
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Investigating IP3 experimentally
- recording calcium levels in permeabilised cells:
- 1) add ATP: Ca sequestered by ER
- 2) Add Ca
- 3) Add IP3 : Ca levels increase again, as Ca is released from ER
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3 main ways of desensitising beta-adrenoceptor
- 1) Uncoupling GPCR and G protein (phosphorylation at GPCR)
- 2) Sequestration of receptors (endocytosis -> destroyed in lysosomes/shuttled back to membrane)
- 3) Downregulation of receptor production (maybe PKA alters mRNA stability?)
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Describe heterologous desensitisation
- Beta2 stimulation produces PKA
- PKA phosphorylates beta 2 receptor at 3rd intracellular loop and C terminal tail (and other similar receptors)
- happens with low [ligand], as is proportional to response
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Describe homologous desensitisation
- ligand bound beta2R phosphorylated by betaARK (adrenoceptor kinase) at C terminal tail
- this increases the affinity of beta-arrestin
- beta-arrestin binding uncouples the receptor
- happens with high [ligand] conc, as it is proportional to occupancy
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4 viral oncogenes thata affect RTKs
useful drug?
- v-erbB: causes a truncated, constitutively active EGFR, found in avian erythroblastosis virus
- c-erbB: overexpressed in squamous cell carcinomas
- v-sis: PDGF (platelet derived growth factor) chain, found in simian sarcoma virus
- c-sis: overexpression is tumorigenic
tyrphostins: inhibit tyrosine kinases, may be useful for controlling neoplastic growth
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Imatinib (Glivec)
Inhibits kinase bcr-abl in chronic myeloid leukaemia
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hsp90
other similar?
- complexes with inactive steroid receptors: needed for glucocorticoids to bind
- hsp70: another essential chaperone
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Different types of sodium channel
- nerves: Nav1.1-1.3
- skeletal muscle: Nav1.4
- cardiac muscle: Nav1.5
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3 reasons why nAChR has been so widely studied
- alpha-bungarotoxin is a high affinity ligand that is used as a competitive antagonist
- receptor structure preserved when solubilised in triton-x 100
- nAChR available from Torpedo electroplaques
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nAChR structure and binding
- 5 subunits: alpha1, alpha2, beta, gamma, delta
- ACh binds between alpha and delta, and alpha and gamma
- each subunit has 4 hydrophobic stretches (M1-M4)
- M2 may form an alpha helix in the membrane, and the 5 M2 regions line the pore of the channel
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Structure of GPCR
- Single subunit
- 7 membrane spanning alpha helices
- Extracellular N terminal tail
- Intracellular C terminal tail
- Ligand binding site within TM helices
- Interact with G proteins with 3rd intracellular loop and C terminal tail
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What experiments found the difference between homologous and heterologous desensitisation?
- Site-directed mutagenesis
- Type A: could not be phosphorylated at PKA site - desensitisation only at high levels of stimulation with isoprenaline
- B: could not be phosphorylated at betaARK site - desensitisation at low (nm) and high (um) levels
- C: could not be phosphorylated at all - no desensitisation
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Structure of receptor tyrosine kinase
- 4 domains: ligand binding, TM, catalytic, autophosphorylation
- Insulin: 2 chains linked as a dimer
- EGF and PDGF: single polypeptide chain
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Structure of steroid receptor
- 3 domains:
- N terminal: transcription activating domain
- DNA binding domain: zinc finger
- Hinge region: nuclear localisation sequence exposed on binding
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