Involvement of the N-terminus of Kir6.2 in coupling to the sulphonylurea receptor

1. ATP-sensitive potassium (K-ATP) channels are. composed of pore-forming K ir6.2 and regulatory SUR subunits. ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction wit h a high-affinity site on SUR1 and a low-affinity site on Kir6.2. MgADP and diazoxide interact with SUR1 to promote channel activity. 2. We examined the effect of N-terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recordin g macroscopic currents in membrane patches excised from Xenopus oocytes exp ressing willd-type or mutant Kir6.2/SUR1. 3. A 14 amino acid N-terminal deletion (Delta N14) did not affect the gatin g, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, K ir6.2 Delta C26, expressed in the absence of SUR1.. Thus, the N-terminal de letion does not alter the intrinsic properties of Kir6.2. 4. When Kir6.2 Delta N14 was coexpressed with SUR1, the resulting K-ATP cha nnels had a higher open probability (P-o = 0.7) and a lower ATP sensitivity (K-i = 196 mu M) than wild-type (Kir6.2/SUR1) channels (P-o = 0.32, K-1 = 28 mu M). High-affinity tolbutamide block was also abolished. 5. Truncation of five or nine amino acids from the N'-terminus of Kir6.2 al so enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high-affinity tolbutamide block, although to a lesser exten t than for the Delta N14 deletion. Site-directed mutagenesis suggests that hydrophobic residues in Kir6.2 may be involved in this effect. 6. The reduced ATP sensitivity of Kir6.2 Delta N14 may be explained by the increased P-o. However, when the P-o was decreased (by ATP), tolbutamide wa s unable to block Kir6.2 Delta N14/SUR1 K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166S/SUR1-K719A,K1385M currents (which in the absence of ATP have a P-o of > 0.8 and are not blocked by tolbutami de). Thus the N-terminus of Kir6.2 may be involved in coupling sulphonylure a binding to SUR1 to closure of the Kir6.2 pore.