Syntaxin-1A Interacts with Distinct Domains within Nucleotide-binding Folds of Sulfonylurea Receptor 1 to Inhibit β-Cell ATP-sensitive Potassium Channels

The ATP-sensitive potassium (K-ATP) channel regulates pancreatic beta-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K-ATP channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K-ATP inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W-A), signature sequence linker, and Walker B (W-B), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W-A and W-B regions of both NBFs. Patch clamp recordings on INS-1 and primary rat beta-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W-B (not NBF1-W-A), NBF2-W-A, and NBF2-W-B. The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W-A (K719M) or NBF2-W-A (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K-ATP channels. These data suggest that NBF1-W-A (Lys-719) and NBF2-W-A (Lys-1385) are critical for Syn-1A-K-ATP channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W-A motifs and sites adjacent to W-B motifs of NBF1 and NBF2 but transduces its inhibitory actions on K-ATP channel activity via some but not all of these NBF domains.