SUR-dependent modulation of KATP channels by an N-terminal KIR6.2 peptide -: Defining intersubunit gating interactions

Ntp and Ctp, synthetic peptides based on the N- and C-terminal sequences of K(IR)6.0, respectively, were used to probe gating of K(IR)6.0/SUR K-ATP channels. Micromolar Ntp dose-dependently increased the mean open channel probability in ligand-free solution (P-O(max)) and attenuated the ATP inhibition of K(IR)6.2/SUR1, but had no effect on homomeric K(IR)6.2 channels. Ntp (up to similar to10(-4) M) did not affect significantly the mean open or "fast," K+ driving force-dependent, intraburst closed times, verifying that Ntp selectively modulates the ratio of mean burst to interburst times. Ctp and Rnp, a randomized Ntp, had no effect, indicating that the effects of Ntp are structure specific. Ntp opened K(IR)6.1/SUR1 channels normally silent in the absence of stimulatory Mg- nucleotide(s) and attenuated the coupling of high-affinity sulfonylurea binding with KATp pore closure. These effects resemble those seen with N-terminal deletions (DeltaN) of K(IR)6.0, and application of Ntp to DeltaNK(ATP) channels decreased their P-O(max) and apparent IC50 for ATP in the absence of Mg2+. The results are consistent with a competition between Ntp and the endogenous N terminus for a site of interaction on the cytoplasmic face of the channel or with partial replacement of the deleted N terminus by Ntp, respectively. The K-IR N terminus and the TMD0-L0 segment of SUR1 are known to control the P-O(max). The L0 linker has been reported to be required for glibenclamide binding, and DeltaNK(IR)6.2/SUR1 channels exhibit reduced labeling of KIR with I-125-azidoglibenclamide, implying that the KIR N terminus and L0 of SUR1 are in proximity. We hypothesize that L0 interacts with the KIR N terminus in ligand-inhibited K-ATP channels and put forward a model, based on the architecture of BtuCD, MsbA, and the KcsA channel, in which TMD0-L0 links the MDR-like core of SUR with the K-IR pore.