Selective Modulation of Neuronal Nicotinic Acetylcholine Receptor Channel Subunits by G sub(o)-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 GTP gamma S 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 GTP gamma S. In inside-out membrane patches, the open probability (NP sub(o)) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mM GTP gamma S relative to control but was unchanged in the presence of GDP beta S. 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 GTP gamma S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G sub(o) alpha or G beta gamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G beta gamma increased the open probability of ACh-activated single-channel currents fivefold, whereas G sub(o) alpha (50 nM) produced no significant increase in NP sub(o). Neuronal nAChR subunits alpha 3- alpha 5 and beta 2 exhibited a positive interaction with G sub(o) alpha and G beta gamma , whereas beta 4 and alpha 7 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.