SK4/IK1 encodes an intermediate conductance, Ca2+ -activated K+ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca2+ -dependent K+ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg2+ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 microM), a PKA inhibitor, to the pipette solution containing ATP/Mg2+ induced a rundown of the Ca2+ -dependent K+ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 microM free Ca2+) containing ATP/Mg2+ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 microM free Ca2+. Forskolin (1 microM), an adenylyl cyclase activator, also increased the currents induced by 0.1 microM free Ca2+. In inside-out macropatches, cytosolic ATP/Mg2+ increased both the maximum current (proportional to the maximum channel activity) and Ca2+ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.