Domain Stability and Functional Analysis at the AD3 Locus of Synaptotagmin 1 C2 Domains

Abstract

pool of dense core granules for exocytosis. Syt-7 also participates in limiting the rate at which fusion pores expand via mechanisms that are not fully clear. All previous imaging studies of Syt-7's actions during exocytosis in chromaffin cells have relied on overexpression of WT or mutant Syt protein. Here, we imaged exocytosis in mouse chromaffin cells without Syt-7 for the first time, so that properties which depend on the protein could be directly determined. Our data show that cells lacking Syt-7 exhibit at least a five-fold difference in fusion efficacy compared to WT cells in response to elevated Kþ depolarization. Lumenal granule cargos are also released at faster rates from Syt-7 knockout (KO) cells, consistent with data from overexpression studies. Next, we measured the responses of WT and KO cells to endogenous secretogoguges of the sympatho-adrenal synapse, acetylcholine (ACh) and pituitary adenylate cyclase-activating peptide (PACAP). Compared to WT cells, ACh-evoked release in KO cells is significantly impaired with respect to granule fusion kinetics and fusion efficacy. PACAP-evoked release, on the hand, is only mildly suppressed. Our data provide evidence for Syt-7's role in two critical aspects of exocytosis -fusion and fusion pore expansion. Moreover, the separable responses of WT and KO cells to endogenous secretogogues suggest that Syt proteins occupy distinct roles in the physiology of the chromaffin cell system. Tandem C2 domain proteins in the synaptotagmin and Doc2 families regulate exocytosis via direct interactions with Ca 2þ and phospholipid bilayers. Synaptotagmin-1 (syt1), a fast-acting, low-affinity Ca 2þ sensor, penetrates membranes upon binding Ca 2þ to trigger synchronous vesicle fusion. Doc2b is a slow-acting, high-affinity Ca 2þ sensor for spontaneous and asynchronous vesicle fusion, but whether it shares a common, penetration-dependent mechanism with syt1 is unknown. In the case of both tandem C2-domain proteins, however, Ca 2þ is thought to be a fundamental requirement for the triggering of exocytosis. Recent work has challenged this model by showing that uncaging of phosphatidylinositol(4,5)-bisphosphate (PIP 2 ) can trigger rapid, syt1dependent exocytosis in the absence of Ca 2þ flux. Here, we reconcile these findings by showing that PIP 2 can drive membrane penetration by syt1 and Doc2b in the absence of Ca 2þ . Ca 2þ -independent penetration of membranes by Doc2b was even more robust than that of syt1, with physiologic levels of PIP 2 driving substantial activation of this sensor. To better define the differences between syt1 and Doc2b, we used the parallax method to measure the membrane penetration depths of syt1 and Doc2b in the presence and absence of Ca 2þ and PIP 2 . We found that, in addition to stimulating Ca 2þ [-independent penetration, PIP 2 exerted different effects on syt1 and Doc2b in the presence of Ca 2þ . While both Ca 2þ sensors underwent Ca 2þ -dependent membrane penetration in the presence of phosphatidylserine (PS), Ca 2þ stimulated only shallow penetra...