M-CURRENTS IN FROG SYMPATHETIC-GANGLION CELLS - MANIPULATION OF MEMBRANE PHOSPHORYLATION

1 The inward current and the M-current (I(M)) suppression produced when muscarine is applied to frog sympathetic ganglion cells was recorded by means of the whole-cell patch-clamp technique. The holding potential was -30 mV and [K+]o was 6 mM. 2 The steady-state I(M) was maintained for at least 20 min when the patch pipette contained neither adenosine 5'-triphosphate (ATP) nor adenosine 3':5'-cyclic monophosphate (cyclic AMP). Inclusion of these substances or the ATP antagonist, beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-MethATP; 1 or 2 nM) (failed to alter the rate of I(M) 'run down'. By contrast, inclusion of adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma-S, 1 or 2 mM) resulted in a 60% reduction of the current within 18 min. 3 Despite the inability of ATP-gamma-S to maintain steady-state I(M), it had no effect on the ability of muscarine (2-100-mu-M) to suppress a constant fraction of the available current. ATP-gamma-S and beta,gamma-MethATP increased the rise time and duration of the response to muscarine. 4 Inclusion of a phosphatase inhibitor, diphosphoglyceric acid (DPG, 1-2.5 mM) or alkaline phosphatase (100-mu-g ml-1) failed to affect the amplitude of muscarinic responses. 5 These results question the role of the phosphorylation and/or dephosphorylation reactions in the transduction mechanism for muscarine-induced I(M) suppression but are consistent with the possibility that M-channels are 'directly coupled' via G-protein to the muscarinic receptor.