Partially active channels produced by PKA site mutation of the cloned renal K1 channel, ROMK2 (kir1.2)

MacGregor, Gordon G., Jason Z. Xu, Carmel M. McNicholas, Gerhard Giebisch, and Steven C. Hebert. Partially active channels produced by PKA site mutation of the cloned renal K1 channel, ROMK2 (kir1.2). Am. J. Physiol. 275 (Renal Physiol. 44): F415–F422, 1998.—The activity of the cloned renal K1 channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert. J. Biol. Chem. 271: 9313–9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability (Po), whereas the NH2-terminal mutation at site S25A showed no change in Po compared with wild-type ROMK2. The decrease in Po for the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was ,66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wildtype ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.