Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by Go-protein subunits.
G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTPgammaS increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTPgammaS. In inside-out membrane patches, the open probability (NP(o)) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mm GTPgammaS relative to control but was unchanged in the presence of GDPbetaS. The modulation of nAChR-mediated whole-cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTPgammaS. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or Gbetagamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nm Gbetagamma increased the open probability of ACh-activated single-channel currents fivefold, whereas G(o)alpha (50 nm) produced no significant increase in NP(o). Neuronal nAChR subunits alpha3-alpha5 and beta2 exhibited a positive interaction with G(o)alpha and Gbetagamma, whereas beta4 and alpha7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.
Fischer
Harald
H
School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Liu
Dong-Mei
DM
Lee
Aven
A
Harries
Janet C
JC
Adams
David J
DJ
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D005726:Ganglia, Parasympathetic / Q000166:cytology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D047468:Immunoprecipitation / Q000379:methods; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008967:Molecular Biology / Q000379:methods; D009474:Neurons / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D009538:Nicotine / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D011336:Probability; D051381:Rats; D011978:Receptors, Nicotinic / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
D011978:Receptors, Nicotinic; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009538:Nicotine; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go