Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines

Abstract

Aims/hypothesis. 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/interpretation. 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. [Diabetologia (2002)

Synaptic transmission at retinal ribbon synapses

“…This isoform substitution may be related to the L-type presynaptic calcium channel used by photoreceptors and bipolar cells. Syntaxin 3 is also present in insulin-secreting β cells where it inhibits L-type Ca 2+ channels and insulin secretion (Kang et al, 2002). The other components of the mininal core fusion complex, SNAP-25 and synaptobrevin, are now believed to be present in retinal ribbon synapses, despite earlier controversies (Von Kriegstein et al, 1999;Sherry et al, 2001Sherry et al, , 2003b.…”

K+Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

“…Essential to this process are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that form the fusion complex that includes syntaxin 1A (syntaxin), SNAP25, and VAMP2 (Bennett et al, 1992a,b;Sollner et al, 1993;Bennett, 1995;Hanson et al, 1997;Hay and Scheller, 1997). Voltage-gated Ca 2ϩ channels, which mediate the influx of Ca 2ϩ that triggers release (Bajjalieh and Scheller, 1995;Bennett, 1995;Sudhof, 1995;Hanson et al, 1997;Chapman, 2002), interact physically with protein components of the fusion complex (for review, see Catterall, 2000;Spafford et al, 2003), and accumulating evidence indicates that these interactions regulate the efficacy of neurotransmitter release (Mochida et al, 1996(Mochida et al, , 1998(Mochida et al, , 2003Rettig et al, 1997;Wiser et al, 1999;Kang et al, 2002;Harkins et al, 2004). Another class of ion channels that influence exocytosis is the voltage-gated K ϩ (Kv) channels, the function of which is thought to be exerted solely and indirectly through their influence on membrane potential (Meir et al, 1999).…”

Transgenic Mouse Overexpressing Syntaxin-1A as a Diabetes Model

“…The voltage-gated K ϩ (K v ) channel, specifically K v 2.1, is then activated, which causes membrane repolarization, resulting in closure of the Ca 2ϩ channel that leads to a cessation of insulin secretion. SNARE proteins, particularly syntaxin-1A (STX-1A), have been shown to directly bind and regulate not only the L-type Ca 2ϩ channels (4,5), but also K ATP (6) and K v 2.1 channels (7,8) of the islet ␤-cells. This led to our broad hypothesis that these SNARE proteins could orchestrate the sequence of ionic and exocytic events leading to insulin secretion.…”