In atrial cells, the open probability of G protein-activated ACh-sensitive K⁺ (K_ACh) channels can be increased approximately fivefold by intracellular ATP (ATP_i). Using inside-out patches, we examined how proteases, changes in intracellular pH, and different anions affect G protein-mediated activation and ATP-induced stimulation of the K_ACh channel. Treatment with trypsin (0.5 mg/ml) removed the GTP dependence of the K_ACh channel and abolished the ATP-induced stimulation. Intracellular GTP activated K_ACh channels at all intracellular pH values tested (6.0-8.0), with the concentration at which half-maximal activation (K_1/2) occurred ranging from 0.3 (pH 8.0) to 6.7 (pH 6.0) µM. However, the ATP_i-induced increase in K_ACh channel activity was inhibited at pH 8.0 (K_1/2 = pH 7.4). All anions tested except sulfate, phosphate, fluoride, and iodide supported GTP-induced activation. Of the anions that supported GTP-induced activation, only citrate blocked the ATP-induced stimulation of the K_ACh channel. These results indicate that the GTP- and ATP-mediated effects on the K_ACh channel use separate signaling pathways. The ATP-mediated effect involves a trypsin- and pH-sensitive mechanism.