H3 Domain of Syntaxin 1A Inhibits KATP Channels by Its Actions on the Sulfonylurea Receptor

The ATP-sensitive potassium (KATP) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the KATP channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K and Ca channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell KATP channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell KATP channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the KATP channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the KATP current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the KATP channels. The Cterminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal HABC domain (Syn-1A-HABC), binds the SUR1 protein of BA8 cells, causing an inhibition of KATP currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis. The pancreatic islet beta cell KATP 1 channel is an octameric complex consisting of four pore-forming Kir6.2 (Kir) subunits and four regulatory SUR1 subunits. The KATP channel functions to link intracellular metabolism, in particular, glucosemediated changes in the ATP/ADP ratio, to the dynamic regulation of cell membrane potential (1). Specifically, an increase in intracellular ATP/ADP ratio in beta cells inhibits KATP channels, which causes membrane depolarization and leads to the opening of voltage-gated Ca channels to trigger exocytosis of the insulin-containing vesicles (1, 2). SUR is a member of the ATP-binding cassette (ABC) superfamily with two cytoplasmic nucleotide-binding folds (NBF-1 and NBF-2), and each NBF contains adenine nucleotide binding Walker A and Walker B motifs (3, 4). The importance of NBFs in regulating KATP channel function has been demonstrated by mutational studies within the NBFs, which abolish the activation of KATP channels by the adenine nucleotides (5) and by metabolic inhibition (6–9). SNARE proteins, which mediate exocytosis, are now known to also regulate a number of ion channels involved in the process of exocytosis. SNARE proteins may therefore serve to orchestrate the sequence of ionic and exocytic events leading to secretion. Indeed, in the neuroendocrine islet beta cells, syntaxin 1A (Syn-1A) and SNAP-25 modulate voltage-gated Ca (10, 11) and voltage-gated K channels (Kv2.1) (12). Recently, we have reported that Syn-1A also inhibits KATP channels in pancreatic islet beta cells, through its actions on the NBF-1 of SUR1 (13). At a resting state, Syn-1A exists in the “closed” configuration in which the N-terminal HABC domain flips over to block the C-terminal H3 domain from undergoing assembly with the cognate SNARE proteins to form the exocytic SNARE complex (14–16). Activation of Syn-1A to an “open” configuration is required to release the inhibitory action of the HABC domain on the H3 domain (17, 18). The H3 domain has been purported to be also the “business end” of Syn-1A, which interacts with the following ion channels: N-type Ca channels (19), cystic fibrosis transmembrane conductance regulator proteins (20), and the epithelial Na channel (21). Taken together, these studies suggest that the H3 domain of Syn-1A might also be the domain that regulates the beta cell KATP channels. In the present study, we examined the actions of the open conformation of Syn-1A and its Syn-1A-H3 domain on SUR1/ * This work is supported by grants from the Juvenile Diabetes Research Foundation (1-2001-521) and Canadian Institutes for Health Research (CIHR MOP-69083) (to H. Y. G.), and the Heart and Stroke Foundation of Ontario (T5343 to R. G. T and H. Y. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § These authors contributed equally to this work. ¶ Supported by fellowships from the Canadian Association of Gastroenterology/Janssen/CIHR. Supported by fellowships from the Canadian Diabetes Association. ‡‡ Supported by a New Investigator Award from the Heart and Stroke Foundation of Canada. §§ To whom correspondence should be addressed: Rm. 7226 Medical Science Bldg., University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1526; Fax: 416-978-8765; E-mail: Herbert.gaisano@ utoronto.ca. 1 The abbreviations used are: KATP, ATP-sensitive K ; SUR, sulfonylurea receptor; ABC, ATP-binding cassette; Syn-1A, syntaxin 1A; wt, wild type; SNAP-25, synaptic proteins of 25-kDa; SNARE, soluble NSF attachment protein receptor; NSF, N-ethylmaleimide-sensitive factor; NBF, nucleotide-binding fold; GST, glutathione S-transferase; HEK, human embryonic kidney; pF, picofarad; I-V, current-voltage. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 51, Issue of December 17, pp. 53259–53265, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.