Abstract
Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Cav). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Cav opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet β-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Cavα1 pore-forming subunits of R-type Cav2.3 and to lesser extent L-type Cavs, while confirming the preferential interactions between Syn-1A with L-type (Cav1.2, Cav1.3) Cavs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Cav2.3, whereas Syn-1A prefers L-type Cavs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Cav channels. Syn-3 KD enhanced Ca2+ currents by 46% attributed mostly to R- and L-type Cavs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Cav activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Cav activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Cavs, but preferring Cav2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS.
Highlights
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including target- (t-) membrane SNAREs (Syntaxins [Syn]) and synaptosomal-associated proteins of 25 kDa (SNAP25) and vesicle-associated membrane proteins (VAMPs), are the fundamental components of the exocytotic machinery required for the docking and fusion of secretory granules (SGs) with the plasma membrane (PM), which have been well studied in neurons [1, 2] and neuroendocrine cells, pancreatic islet β-cells [3,4,5]. t-SNAREs
glucagon-like peptide-1 (GLP-1)/high glucose stimulation caused a large increase in the amount of Cav2.3 co-precipitated, a more moderate increase in Cav1.2 co-precipitated, and no significant increase in Cav1.3 co-precipitated (1.9% to 2.0%)
More work will be required to elucidate the putative functional voltage-gated calcium channels (Cav)-binding domain(s) within the Syn-3 cytoplasmic domain. These results demonstrate that Syn-3, via its cytoplasmic domain, preferentially binds and regulates R-type/Cav2.3 α1 subunit
Summary
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including target- (t-) membrane SNAREs (Syntaxins [Syn]) and synaptosomal-associated proteins of 25 kDa (SNAP25) and vesicle-associated membrane proteins (VAMPs), are the fundamental components of the exocytotic machinery required for the docking and fusion of secretory granules (SGs) with the plasma membrane (PM), which have been well studied in neurons [1, 2] and neuroendocrine cells, pancreatic islet β-cells [3,4,5]. t-SNAREsPLOS ONE | DOI:10.1371/journal.pone.0147862 February 5, 2016Syntaxin-3/Cav2.3 Forms the Excitosome for Newcomer Granule. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including target- (t-) membrane SNAREs (Syntaxins [Syn]) and synaptosomal-associated proteins of 25 kDa (SNAP25) and vesicle-associated membrane proteins (VAMPs), are the fundamental components of the exocytotic machinery required for the docking and fusion of secretory granules (SGs) with the plasma membrane (PM), which have been well studied in neurons [1, 2] and neuroendocrine cells, pancreatic islet β-cells [3,4,5]. Syntaxin-3/Cav2.3 Forms the Excitosome for Newcomer Granule
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