title: Syntaxin-1A interacts with distinct domains within nucleotide-binding folds of sulfonylurea receptor 1 to inhibit beta-cell ATP-sensitive potassium channels.
abstract: The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-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 β-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.
authors.affiliation: Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Nathan
authors.initials: N
authors.lastname: Chang
authors.forename: Tao
authors.initials: T
authors.lastname: Liang
authors.forename: Xianguang
authors.initials: X
authors.lastname: Lin
authors.forename: Youhou
authors.initials: Y
authors.lastname: Kang
authors.forename: Huanli
authors.initials: H
authors.lastname: Xie
authors.forename: Zhong-Ping
authors.initials: ZP
authors.lastname: Feng
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D064233:Sulfonylurea Receptors; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C577507:ABCC8 protein, human; D018528:ATP-Binding Cassette Transporters; C577509:Abcc8 protein, rat; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D050827:Syntaxin 1