H3 domain of syntaxin 1A inhibits KATP channels by its actions on the sulfonylurea receptor 1 nucleotide-binding folds-1 and -2.
The ATP-sensitive potassium (K(ATP)) 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 K(ATP) 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(2+) channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K(ATP) 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 K(ATP) 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 K(ATP) 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 K(ATP) 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 K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) 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.
Cui
Ningren
N
Department of Medicine, University of Toronto, Toronto M5S 1A8, Canada.
Kang
Youhou
Y
He
Yan
Y
Leung
Yuk-Man
YM
Xie
Huanli
H
Pasyk
Ewa A
EA
Gao
Xiaodong
X
Sheu
Laura
L
Hansen
John B
JB
Wahl
Philip
P
Tsushima
Robert G
RG
Gaisano
Herbert Y
HY
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D006657:Histones / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011955:Receptors, Drug / Q000737:chemistry* / Q000378:metabolism; D064233:Sulfonylurea Receptors; D050827:Syntaxin 1; D014162:Transfection
C577507:ABCC8 protein, human; D018528:ATP-Binding Cassette Transporters; C577509:Abcc8 protein, rat; D000954:Antigens, Surface; D006657:Histones; D007328:Insulin; D009419:Nerve Tissue Proteins; D009711:Nucleotides; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; C494212:STX1A protein, human; C494213:Stx1a protein, rat; D064233:Sulfonylurea Receptors; D050827:Syntaxin 1; D005982:Glutathione Transferase; D011188:Potassium