title: Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines.
abstract: OBJECTIVE
  Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, -3 and -4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process.
  
  
  METHODS
  We examined the following effects of Syn-1, -2, -3 and -4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The L-type Ca(2+) channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter.
  
  
  RESULTS
  Syn-1A or -3, but not Syn-2 or -4 overexpression, inhibited K(+)-induced insulin release as determined by RIA (49.7 +/- 5.5 % and 49.1 +/- 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 +/- 21.0 % and 58.0 +/- 13.2 %, respectively). Overexpressed Syn-1A and -3, but not Syn-2, inhibited Ca(2+) channel current amplitude by 39.5 +/- 11.6 % and 52.7 +/- 6.0 %, respectively. Of note, overexpression of Syn-1A and -3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 +/- 4.2 % and 31.8 +/- 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 +/- 4.2 % and 25.7 +/- 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 +/- 9.15 % and 39.0 +/- 6.8 %, respectively).
  
  
  CONCLUSIONS
  These results demonstrate that Syn-1A and -3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and -4 do not inhibit the insulin secretory process.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Kang
authors.forename: X
authors.initials: X
authors.lastname: Huang
authors.forename: E A
authors.initials: EA
authors.lastname: Pasyk
authors.forename: J
authors.initials: J
authors.lastname: Ji
authors.forename: G G
authors.initials: GG
authors.lastname: Holz
authors.forename: M B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: R G
authors.initials: RG
authors.lastname: Tsushima
authors.forename: H Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002460:Cell Line; D017931:DNA Primers; D005089:Exocytosis / Q000187:drug effects; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics*; D018408:Patch-Clamp Techniques; D011401:Promoter Regions, Genetic; D050765:Qa-SNARE Proteins; D011863:Radioimmunoassay; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050827:Syntaxin 1; D014162:Transfection
chemical_list: D000954:Antigens, Surface; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D017931:DNA Primers; D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; C494214:Stx1a protein, mouse; C494213:Stx1a protein, rat; D050827:Syntaxin 1