Synaptotagmin‐7 enhances calcium‐sensing of chromaffin cell granules and slows discharge of granule cargos
Bendahmane, Mounir; Morales, Alina; Kreutzberger, Alex J. B.; Schenk, Noah A.; Mohan, Ramkumar; Bakshi, Shreeya; Philippe, Julie M.; Zhang, Shuang; Kiessling, Volker; Tamm, Lukas K.; Giovannucci, David R.; Jenkins, Paul M.; Anantharam, Arun
2020-09
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Bendahmane, Mounir; Morales, Alina; Kreutzberger, Alex J. B.; Schenk, Noah A.; Mohan, Ramkumar; Bakshi, Shreeya; Philippe, Julie M.; Zhang, Shuang; Kiessling, Volker; Tamm, Lukas K.; Giovannucci, David R.; Jenkins, Paul M.; Anantharam, Arun (2020). "Synaptotagmin‐7 enhances calcium‐sensing of chromaffin cell granules and slows discharge of granule cargos." Journal of Neurochemistry 154(6): 598-617.
Abstract
Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.Syt‐7 is a high‐affinity calcium sensor expressed on chromaffin cell dense core granules. The purpose of this study was to assess the role of Syt‐7 in regulating the secretory response to cholinergic stimulation. Acetylcholine elicits secretion by elevating cytosolic calcium. The calcium sensitivity of exocytosis in cells lacking Syt‐7 is impaired. Cells that lack Syt‐7 also release peptide hormones at faster rates, implicating a role for Syt‐7 in regulating the exocytotic fusion pore. These data demonstrate that Syt‐7 has an important role in triggering exocytosis in cells and is likely to play a role in controlling hormone output, in situ.Publisher
Wiley Periodicals, Inc.
ISSN
0022-3042 1471-4159
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