Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: Interactions between SUR1 and positively charged domains on Kir6.2

K-ATP channels, comprised of the pore-forming protein Kir-6.x and the sulfo nylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interacti ons, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP., on channel regulati on by adenine nucleotides and the sulfonylurea glyburide. Our data show tha t the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburid e. These results suggest that R176 and R177 are required for functional cou pling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K-AT P channel. In contrast, the R301C and R314C mutants, which are also "PIP2-i nsensitive," remained sensitive to stimulation by MgADP in the absence of A TP and were inhibited by glyburide. Based on these findings, as well as pre vious data, we propose a model of the K-ATP channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that int eracts with NBF1 bound to ATP on SUR1, promoting channel opening by counter acting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the abse nce of ATP, since K-ATP channels are not blocked by ATP, they do not requir e the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence o f ATP. The region delimited by R301 and R314 is not involved in the interac tion with NBF1 or NBF2, but confers additional PIP2 sensitivity.