Atrial natriuretic peptide, acting through its second messenger guanosine 3',5'-cyclic monophosphate (cGMP), suppresses Na+ absorption across the renal inner-medullary collecting duct and increases urinary Na+ excretion. Patch clamp studies show that cGMP reduces Na+ absorption by inhibiting an amiloride-sensitive cation channel in the apical membrane. We have now examined, using the patch clamp technique, the molecular mechanisms of cGMP inhibition. Cyclic GMP directly and specifically reduced the probability of a single channel being open (open probability, Po) by 39% (inhibition constant, Ki = 7.6 x 10(-7) M) by a phosphorylation-independent mechanism. Cyclic GMP also inhibited the channel by activating cGMP-dependent protein kinase (cGMP-kinase). Exogenous cGMP-kinase completely inhibited the channel by a phosphorylation-dependent mechanism. Activation of a pertussis toxin-sensitive G protein by GTP-gamma-S blocked cGMP-kinase inhibition of the channel. By contrast, cGMP-kinase inhibition of Po was completely reversed by GTP-gamma-S. Taken together with the results of a previous study showing that a G protein activates the cation channel, these data indicate that cGMP-kinase and a G protein sequentially regulate the cation channel. Our results show that atrial natriuretic peptide, acting through cGMP, inhibits Na+ absorption across the inner-medullary collecting duct by a dual mechanism, and that cGMP-kinase inhibits the channel by a pathway involving a G protein.