Jgp_201711757 1..18

Regulated exocytosis in chromaffin cells is triggered by membrane depolarization and subsequent Ca influx through voltage-gated channels. The level of Ca accumulation is commensurate with the strength of stimulation (Douglas and Rubin, 1961; Neher and Augustine, 1992; Fulop and Smith, 2007; de Diego et al., 2008). Ca drives exocytosis through the Ca-binding synaptotagmin (Syt) protein family (Brose et al., 1992; Voets et al., 2001a; Schonn et al., 2008). The Syt protein family includes 17 isoforms, but only two of these isoforms (Syt-1 and Syt-7) are known to be expressed on chromaffin cell dense core granules (Schonn et al., 2008). Both Syt isoforms harbor an N-terminal transmembrane domain that extends into the lumen of the chromaffin granule, followed by two cytosolic C2 domains (C2A and C2B) connected by a short linker region (Perin et al., 1990, 1991; Chapman, 2002). The Caand membrane-binding properties of these isoforms are determined primarily by the amino acid sequence within the tandem C2 domains (Sutton et al., 1995; Ubach et al., 1998; Fernandez et al., 2001). Biochemical studies have established several differences in how these isoforms respond to Ca. For example, Syt-7 is capable of binding a total of six Ca ions, while Syt-1 can bind to only five (Südhof and Rizo, 1996; Ubach et al., 1998). Although both proteins bind membranes in a Ca-dependent manner, Syt-7 does so with a 10-fold higher sensitivity for Ca ions compared with Syt-1 (Sugita et al., 2002; Bhalla et al., 2005). The notion that granule or vesicle proteins may confer spatiotemporal heterogeneity to fusion events has recently become more widely appreciated. At synapses, there is evidence that vesicle-associated membrane protein/synaptobrevin isoforms may act to sort vesicles into synchronous, asynchronous, and spontaneously fusing populations (Raingo et al., 2012; Bal et al., 2013; Crawford and Kavalali, 2015). Syt isoforms may serve similar Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca. Upon introduction of Ca into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca for activation. At Ca concentrations above 30 μM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca sensitivities. Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules