Ca2+-binding mutant C2AB-4D/N (36) exhibited a similarly small amount of binding to both Syns in absence or presence of Ca2+, indicating some degree of Ca2+-impartial interaction. 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), BRD-6929 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 -cells than its previously purported role in exocytotic fusion per se. Introduction In exocytosis, soluble N-ethylmaleimideCsensitive factor attachment protein receptor (SNARE) proteins on secretory granules (SGs) and plasma membrane (PM) are assembled into a complex assisted by accessory proteins to hold SGs close to the PM (1). Synaptotagmins (Syts) are major Ca2+ sensor proteins attached to SNARE complexes, which, when bound to Ca2+, trigger SNARE complexCmediated exocytotic fusion (1C3). In pancreatic islet -cells, recruitment and exocytosis of insulin SGs exhibit a biphasic glucose-stimulated insulin secretory (GSIS) pattern consisting of a robust first phase followed by a sustained second phase (4). Insulin SGs undergo several modes of exocytosis that underlie each of the two phases of GSIS. In the first mode, SGs are recruited to dock around the PM followed by priming; then these SGs sit on the PM for an indefinite time (hence being termed predocked SGs) until Ca2+-brought on exocytosis. Predocked SGs are purported to form the readily releasable pool (RRP) that is a major contributor to first-phase GSIS (5). In the second mode, insulin SGs are mobilized from a reserve pool (RP) in the -cell interior to the PM to undergo fusion with only a short BRD-6929 period or almost BRD-6929 no docking time at the PM. These SGs, coined newcomer SGs, are responsible for almost all of second-phase GSIS and a substantial proportion of first-phase GSIS (6C11). First-phase release of predocked SGs requires high intracellular calcium concentration (half-maximal effective concentration value of 10 mol/L) (12,13), whereas second-phase secretion is usually operated under lower intracellular calcium concentration, the latter indicating a different pool of SGs (newcomer SGs) that exhibit very high Ca2+ affinity (half-maximal effective concentration value of a few micromoles) (14). Taken together, these reports suggest the involvement of different SNARE complexes and cognate Ca2+ sensors for predocked and newcomer SGs (7). We decided the SNARE fusion machinery of newcomer SGs, which consists of Syn-3, VAMP8, and accessory priming factor Munc18b (15C17). This is distinct from the fusion machinery of predocked SGs (i.e., Syn-1A, VAMP2, and Munc18a) (5C7). However, what the putative Ca2+ sensor(s) is for predocked and newcomer SGs has not been definitively exhibited. Syts constitute a family of transmembrane proteins with two KLF4 cytoplasmic C2 domains (C2A and C2B) that exhibit distinct binding properties to effectors Ca2+, SNARE proteins, and phospholipids (2,3). Of 17 mammalian Syt isoforms, several have been postulated to regulate insulin SG exocytosis (18), of which Syt-7 appears to be the putative one (19C22). This was definitively shown by genetic deletion of Syt-7 in mice, which reduced biphasic GSIS (21,22). However, in those studies (21,22), the precise role of Syt-7 in insulin SG exocytosis per se was not elucidated. It was assumed that Syt-7s major action is to drive BL21 (DE3) for protein expression. GST-fusion proteins were then purified using glutathione-Sepharose beads. HEK293 cells were transfected with pcDNA3.1-Syn-1A or pcDNA3.1-Syn-3 plasmids using Lipofectamine 2000 (Invitrogen); 48-h posttransfected cells were then washed with PBS and lysed in binding buffer (20 mmol/L HEPES, 100 BRD-6929 mmol/L KCl, 1.5% Triton X-100, 1 g/ml leupeptin, and 10 g/ml aprotinin [pH 7.4]) at 4C. For Ca2+ dependence.
Ca2+-binding mutant C2AB-4D/N (36) exhibited a similarly small amount of binding to both Syns in absence or presence of Ca2+, indicating some degree of Ca2+-impartial interaction
