PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.
ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.
Béguin
P
P
Department of Molecular Medicine, Chiba University Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan.
Nagashima
K
K
Nishimura
M
M
Gonoi
T
T
Seino
S
S
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism; D064233:Sulfonylurea Receptors; D013997:Time Factors; D014981:Xenopus / Q000196:embryology
C577507:ABCC8 protein, human; C577510:ABCC9 protein, human; D018528:ATP-Binding Cassette Transporters; C577509:Abcc8 protein, rat; C577512:Abcc9 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase