M-currents in frog sympathetic ganglion cells: manipulation of membrane phosphorylation.
1. The inward current and the M-current (IM) 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 IM 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 IM '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 IM, it had no effect on the ability of muscarine (2-100 microM) 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 micrograms 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 IM suppression but are consistent with the possibility that M-channels are 'directly coupled' via G-protein to the muscarinic receptor.
Department of Pharmacology, University of Alberta, Edmonton, Canada.
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D000818:Animals; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D005728:Ganglia, Sympathetic / Q000502:physiology*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000502:physiology*; D008566:Membranes / Q000378:metabolism; D009116:Muscarine / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation; D011894:Rana pipiens; D011976:Receptors, Muscarinic / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology
D004163:Diphosphoglyceric Acids; D007473:Ion Channels; D009711:Nucleotides; D011976:Receptors, Muscarinic; D009116:Muscarine