MOLECULAR ANALYSIS OF ATP-SENSITIVE K-CHANNEL GATING AND IMPLICATIONSFOR CHANNEL INHIBITION BY ATP

The beta cell K-ATP channel is an octameric complex of four pore-formi ng subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2 Delta C26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit i s primarily responsible for mediating ATP inhibition. We show here tha t mutation of C166, which lies at the cytosolic end of the second tran smembrane domain, to serine (C166S) increases the open probability of Kir6.2 Delta C26 approximately sevenfold by reducing the time the chan nel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2 Delta C26 containing the C166S mutation shows a mar kedly reduced ATP sensitivity: the K-i is reduced from 175 mu M to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simul taneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduc ed in channels composed of SUR1 and Kir6.2 carrying the C166S mutation . Our results are consistent with the idea that C166 plays a role in t he intrinsic gating of the channel, possibly by influencing a gate loc ated at the intracellular end of the pore. Kinetic analysis suggests t hat the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.