PKA-mediated phosphorylation of the human K-ATP channel: separate roles ofKir6.2 and SUR1 subunit phosphorylation

ATP-sensitive potassium (K-ATP) channels play important roles in many cellu lar functions such as hormone secretion and excitability of muscles and neu rons. Classical ATP-sensitive potassium (K-ATP) channels are heteromultimer ic membrane proteins comprising the pore-forming Kir6.2 subunits and the su lfonylurea receptor subunits (SUR1 or SUR2), The molecular mechanism by whi ch hormones and neurotransmitters modulate K-ATP channels via protein kinas e A (PKA) is poorly understood. We mutated the PKA consensus sequences of t he human SUR1 and Kir6.2. subunits and tested their phosphorylation capacit ies in Xenopus oocyte homogenates and in intact cells. We identified the si tes responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571), Kir6.2 can be phosphorylated at its PKA phosphorylation s ite in intact cells after G-protein (Gs)-coupled receptor or direct PKA sti mulation. While the phosphorylation of Kir6.2 increases channel activity, t he phosphorylation of SUR1 contributes to the basal channel properties by d ecreasing burst duration, interburst interval and open probability, and als o increasing the number of functional channels at the cell surface, Moreove r, the effect of PKA could be mimicked by introducing negative charges in t he PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K-ATP channel, and may explain the mechanism by which Gs-cou pled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally di stinct roles of the phosphorylation of the different subunits.