Syntaxin 1 interacts with the L(D) subtype of voltage-gated Ca(2+) channels in pancreatic beta cells.
Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) 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 K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.
Yang
S N
SN
The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Larsson
O
O
Bränström
R
R
Bertorello
A M
AM
Leibiger
B
B
Leibiger
I B
IB
Moede
T
T
Köhler
M
M
Meister
B
B
Berggren
P O
PO
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D020746:Calcium Channels, L-Type; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D005089:Exocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins; D007515:Islets of Langerhans / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D008820:Mice, Obese; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050827:Syntaxin 1; D014404:Tubulin / Q000276:immunology / Q000502:physiology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C494214:Stx1a protein, mouse; D050827:Syntaxin 1; D014404:Tubulin; D049452:Green Fluorescent Proteins; D002118:Calcium