Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet β-Cells

Abstract

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7’s precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus–short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca2+-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se.

Recent new insights into the role of SNARE and associated proteins in insulin granule exocytosis

“…This is in addition to Syn‐1A's known actions on predocked SG that contributes to first‐phase GSIS . This was also the case with syt‐7 depletion in human β‐cells . In fact, this role of syt‐7 in the replenishment of SGs from the β‐cell interior to the PM after glucose‐stimulated emptying of releasable pools superseded its function on the predock and newcomer SG SNARE fusion complexes at the PM .…”

“…The quaternary SM/SNARE complex forms a molecular zipper structure that is only partially zipped up, as it is clamped by Munc18a and synaptotagmin (syt), the latter serving as the primary Ca 2+ sensor . Syt‐7 was found to be the putative Ca 2+ sensor for β‐cells . The current thinking is that Ca 2+ ’s action on syt (probably including syt‐7) unclamps the SNARE complex to complete its zippering up, which induces mixing of the lipid bilayers of the SG and PM that culminates in the formation of a fusion pore for the SG contents to empty into the cell exterior .…”

“…Ca 2+ sensors . Syt‐7 is also the Ca 2+ sensor for newcomer SGs as syt‐7 could also bind Syn‐3 in addition to Syn‐1A . Whether the other Ca 2+ sensors (Doc2, CAPS) and fusion clamps (complexin, tomosyn) discussed above for predocked SG fusion can also influence newcomer SG fusion requires further study.…”

MiR‐335 overexpression impairs insulin secretion through defective priming of insulin vesicles

“…; Dolai et al. ). Indeed, granule docking coincides with the formation of clusters containing STXBP1 (Gandasi and Barg ) and the physiological importance of docking in insulin secretion was shown in the diabetic GK‐rat (Ohara‐Imaizumi et al.…”

“…STXBP1 binds to the folded conformation of STX1A during docking (Han et al 2011), and subsequently dissociates from STX1A upon Ca 2+ influx to allow formation of the SNARE complex (Tomas et al 2008) in the priming step (Gulyas-Kovacs et al 2007). In addition, studies have implicated STXBP1, as well as SNAP25 and the synaptotagmin isoforms 1 and 7, in the recruitment of granules to the membrane (Gulyas-Kovacs et al 2007;Ohara-Imaizumi et al 2007;Tomas et al 2008;de Wit et al 2009;Dolai et al 2016). Indeed, granule docking coincides with the formation of clusters containing STXBP1 (Gandasi and Barg 2014) and the physiological importance of docking in insulin secretion was shown in the diabetic GK-rat (Ohara-Imaizumi et al 2004a).…”