Syntaxin 1 interacts with the L _D subtype of voltage-gated Ca ²⁺ channels in pancreatic β cells

Abstract

Interaction of syntaxin 1 with the ␣ 1D subunit of the voltage-gated L type Ca 2؉ channel was investigated in the pancreatic ␤ cell. Coexpression of the enhanced green f luorescent protein-linked ␣ 1D subunit with the enhanced blue f luorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the ␣ 1D subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the ␣ 1D subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin We now show that syntaxin 1 colocalizes and associates with the ␣ 1D subunit of the voltage-gated L type Ca 2ϩ channel and thereby modulates not only Ca 2ϩ channel activity but also insulin release in a Ca 2ϩ -dependent manner. MATERIALS AND METHODSPreparation of Islets and Single ␤ cells. Islets of Langerhans and single pancreatic ␤ cells were isolated from adult obese mice (gene symbol ob͞ob) as described previously (11).Coexpression of ␣ 1D Subunit-Enhanced Green Fluorescent Protein (EGFP) and Enhanced Blue Fluorescent Protein (EBFP)-Syntaxin 1 and Fluorescence Microscopy. The cDNA for hamster ␣ 1D3a (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 ␣ 1D3a 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 SmaI͞XbaI-opened pCMV EBFP0. Cultured single pancreatic ␤ cells were cotransfected with pCMV ␣ 1D3a EGFP and pCMV EBFPsyntaxin 1A overnight by the lipofectamine technique. The cotransfected cells were monitored for EGFP͞EBFP fluorescence 48 h after the start of transfection by using a Leica Fluovert FU microscope (Leica, Deerfield, IL) and PL Fluotar 100͞1.32 oil lens (Leitz) equipped with a LSR AstroCam TE3͞A͞S charge-coupled device camera (LSR, Cambridge, U.K.). Filter settings for EGFP fluorescence measurements were: excitation, HQ470͞40; dichroic mirror, Q495LP; and emission, HQ525͞50. Filter settings for EBFP fluorescence measurements were: excitation, D399͞22; dichroic mirror, 420DLCP; and emission, E430LP. Colocalization of ␣ 1D3a 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 ...

Newcomer insulin secretory granules as a highly calcium-sensitive pool

“…This agrees with the observation that second-phase secretion from newcomers occurs away from syntaxin clusters (14), which are known to be colocated with Ca 2ϩ channels (39).…”

“…In addition, first-phase secretion from wild-type cells has been found to occur mainly from previously docked granules at Synt1A-rich locations, whereas second-phase secretion is mainly due to newcomer granules fusing away from Synt1A clusters (14). Because syntaxin-1 and L-type Ca 2ϩ channels colocalize (39), the results of Ohara-Imaizumi et al (14) show that first-phase secretion takes place mostly at L-type Ca 2ϩ channels, whereas second-phase fusion events occur away from L-type channels, in accordance with Ca 2ϩ -channel knockout experiments (10,11).…”

Functional Interaction of Auxiliary Subunits and Synaptic Proteins With CaV1.3 May Impart Hair Cell Ca2+Current Properties

“…Localization of Ca 2ϩ channels and syntaxin at the basal pole of hair cells with anti-Ca V 1.3␣ 1 and anti-syntaxin revealed that the two proteins are expressed closely together (Fig. 3), in accordance with previous co-immunoprecipitation and fluorescent-tagged ␣-subunit experiments that demonstrated the association of syntaxin 1A with Ca V 2.2 and Ca V 1.3 channels (Bennett et al 1992;Yang et al 1999). The present findings show that in a heterologous expression system, the association of the ␣, ␤, and ␣ 2 ␦ subunits and synaptic proteins approximate the voltage dependence and inactivation kinetics of the native properties of the Ca v 1.3␣ 1 in hair cells.…”

Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents

“…Islets of these animals express significantly less exocytotic proteins involved in SNARE (soluble N-ethylmaleimide attachment receptor) complex formation because of impaired FoxO1 transcriptional activity 42 . One of these proteins regulated by FoxO1, Syntaxin1A, interacts with the a1D subunit of the L-type voltage-gated Ca 2 þ channel 43 and is implicated in SNARE formation and insulin release 44 . An orthologue in C. elegans is regulated by daf-19, a transcription factor that is expressed in all ciliated sensory neurons 45,46 .…”