Syntaxin 1 interacts with the LD subtype of voltage-gated Ca21 channels in pancreatic b cells

Interaction of syntaxin 1 with the a1D subunit of the voltage-gated L type Ca21 channel was investigated in the pancreatic b cell. Coexpression of the enhanced green fluorescent protein-linked a1D subunit with the enhanced blue f luorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the a1D subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the a1D subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca21 channel activity because there was a faster Ca21 current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K1 or exposure to high Ca21. Exchanging Ca21 for Ba21 abolished the effect of antisyntaxin 1 on both Ca21 channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca21-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure–function relationship between the a1D subunit of the L type Ca21 channel and the exocytotic machinery in the pancreatic b cell. Syntaxin 1 plays a central role in exocytosis from pancreatic b cells (1–3). Thus, it has been demonstrated that exposure of permeabilized mouse pancreatic b cells to antibodies against syntaxin 1 significantly inhibits Ca21-induced insulin secretion (1). Furthermore, synthetic peptides, corresponding to the H3 region of the carboxyl-terminal domain of syntaxin 1, markedly inhibit Ca21-dependent insulin secretion from permeabilized pancreatic b cells (2). In neurons, syntaxin interacts not only with other synaptic proteins but also with voltage-dependent Ca21 channels (4, 5). Several studies indicate that the interaction between syntaxin and voltage-dependent Ca21 channels results in a functional modulation of the Ca21 current. Thus, electrophysiological studies have shown that syntaxin modulates N, LC, and Q type Ca21 currents in Xenopus oocytes, coexpressing voltage-gated Ca21 channels and syntaxin (6–8). It also has been reported that intracellular application of a synthetic peptide fragment of the a1C subunit diminished exocytosis from the pancreatic b cell via interference with the interaction of the L type Ca21 channel with the exocytotic machinery. However, analysis of mRNAs encoding the a1C and a1D subunits implies that the Ca21-conducting subunits of the L type Ca21 channel in the pancreatic b cell mainly consist of the a1D subunit (9, 10). We now show that syntaxin 1 colocalizes and associates with the a1D subunit of the voltage-gated L type Ca21 channel and thereby modulates not only Ca21 channel activity but also insulin release in a Ca21-dependent manner. MATERIALS AND METHODS Preparation of Islets and Single b cells. Islets of Langerhans and single pancreatic b cells were isolated from adult obese mice (gene symbol obyob) as described previously (11). Coexpression of a1D Subunit–Enhanced Green Fluorescent Protein (EGFP) and Enhanced Blue Fluorescent Protein (EBFP)–Syntaxin 1 and Fluorescence Microscopy. The cDNA for hamster a1D3a (provided by J. Dillon, New England Medical Center, Boston) was flanked with the human cytomegalovirus (CMV) promoter and the bovine growth hormone poly(A) site to enable expression in mammalian cells. A HindIII site was introduced by site-directed mutagenesis at the nucleotides coding for the last amino acid and the stop codon, which allowed in-frame fusion with the EGFP cDNA and generation of pCMV a1D3a EGFP. To create pCMV EBFPsyntaxin 1A, a SmaI site was introduced into the EBFP cDNA at the nucleotides coding for Asp-235 and Glu-237, generating pCMV EBFP0. The cDNA for rat syntaxin 1A (gift from R. H. Scheller, Stanford University, Stanford, CA) was removed from pRcCMV syntaxin 1A by digestion with EcoRV and XbaI and fused in-frame with the EBFP cDNA of the SmaIyXbaIopened pCMV EBFP0. Cultured single pancreatic b cells were cotransfected with pCMV a1D3a EGFP and pCMV EBFPsyntaxin 1A overnight by the lipofectamine technique. The cotransfected cells were monitored for EGFPyEBFP fluorescence 48 h after the start of transfection by using a Leica Fluovert FU microscope (Leica, Deerfield, IL) and PL Fluotar 100y1.32 oil lens (Leitz) equipped with a LSR AstroCam TE3yAyS charge-coupled device camera (LSR, Cambridge, U.K.). Filter settings for EGFP fluorescence measurements were: excitation, HQ470y40; dichroic mirror, Q495LP; and emission, HQ525y50. Filter settings for EBFP fluorescence measurements were: excitation, D399y22; dichroic mirror, 420DLCP; and emission, E430LP. Colocalization of a1D3a EGFP and EBFP-syntaxin 1A was studied by applying the deconvolution method on a stack of 15 images (200-nm vertical distance) using the iterative Tikhonov–Miller restoration procedure (Huygens System 2; Scientific Volume Imaging, Hilversum, The Netherlands). Density Gradient Subcellular Fractionation. Subcellular fractionation, using a linear sucrose gradient, was performed as described previously (12). Gel Electrophoresis and Western Blot Analysis. Proteins (90 mgyslot) from individual fractions were separated in discontinuous gels consisting of a stacking gel (3% acrylamide) and a separating gel, SDS-polyacrylamide gradient gel (6.6– 13.3% acrylamide). The separated proteins then were electroblotted to hydrophobic polyvinylidene difluoride membrane (Hybond-P). The blots were blocked by incubation for 2 h with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: EBFP, enhanced blue fluorescent protein; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; CMV, cytomegalovirus; 5-HTP, 5-hydroxy-DL-tryptophan. †To whom reprint requests should be addressed at: Karolinska Hospital (L1:02), S-171 76 Stockholm, Sweden. E-mail: [email protected].