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Complexin II Plays a Positive Role in Ca -Triggered Exocytosis By

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Complexin II Plays a Positive Role in Ca -Triggered Exocytosis By Complexin II plays a positive role in Ca2؉-triggered exocytosis by facilitating vesicle priming Haijiang Caia,1, Kerstin Reimb, Frederique Varoqueauxb, Sompol Tapechuma,2, Kerstin Hillc,3, Jakob B. Sørensenc, Nils Broseb, and Robert H. Chowa,4 aDepartment of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA 90089; bDepartment of Molecular Neurobiology and Center for the Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, D-37075 Go¨ttingen, Germany; and cDepartment of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Go¨ttingen, Germany Communicated by Clay M. Armstrong, University of Pennsylvania, Philadelphia, PA, October 10, 2008 (received for review August 3, 2008) SNARE-mediated exocytosis is a multistage process central to II antibody into Aplysia buccal ganglia neurons stimulated neuro- synaptic transmission and hormone release. Complexins (CPXs) are transmitter release (14), and overexpression of CPX I or CPX II in small proteins that bind very rapidly and with a high affinity to the PC12 cells was reported to suppress acetylcholine (ACh) release SNARE core complex, where they have been proposed recently to (15). More recent studies showed that cell–cell fusion of cells that inhibit exocytosis by clamping the complex and inhibiting mem- express SNAREs facing extracellularly is inhibited by CPX (16–18) brane fusion. However, several other studies also suggest that and that overexpression of a synaptobrevin CPX fusion protein, CPXs are positive regulators of neurotransmitter release. Thus, which is thought to cause high presynaptic levels of CPX, inhibits whether CPXs are positive or negative regulators of exocytosis is synaptic transmission (19). The inhibitory role of CPX was further not known, much less the stage in the vesicle life cycle at which supported by the result that spontaneous release was increased in they function. Here, we systematically dissect the vesicle stages the Drosophila neuromuscular junction where CPX was knocked leading up to exocytosis using a knockout-rescue strategy in a out (20). In addition, data on bovine chromaffin cells overexpress- mammalian model system. We show that adrenal chromaffin cells ing CPX II were interpreted to indicate a role of CPXs in the from CPX II knockout mice exhibit markedly diminished releasable regulation of fusion pore dynamics (21). Taken together, these data vesicle pools (comprising the readily and slowly releasable pools), led to the notion that CPXs play an inhibitory role in fusion by while showing no change in the kinetics of fusion pore dilation or clamping SNARE complexes. morphological vesicle docking. Overexpression of WT CPX II—but Other studies, however, indicate that CPXs play a positive role in not of SNARE-binding-deficient mutants—restores the size of the regulated exocytosis. In pancreatic ␤-cells, insulin secretion is the releasable pools in knockout cells, and in WT cells it markedly enhanced by overexpressing CPX I, and decreased by silencing CPX enlarges them. Our results show that CPXs regulate the size of the I expression with RNA interference (22). In mast cells, a knock- ؉ primed vesicle pools and have a positive role in Ca2 -triggered down of CPX II also led to suppressed secretion (23). CPX I/II exocytosis. double knockout neurons displayed dramatically reduced neuro- transmission, associated with a change in the apparent Ca2ϩ chromaffin cell ͉ large dense core vesicle ͉ SNARE complex sensitivity of exocytosis (24). And distinct domains of CPX are recently proposed to have different functions (25). Thus, the role of roperly regulated exocytosis is important for the release of CPX remains to be clarified (26). Pneurotransmitters and hormones from membrane-delimited For the present study, we used mouse adrenal chromaffin cells, vesicles. Secretory vesicles must dock at the plasma membrane and a well-characterized and extensively used model system for studying then undergo priming or maturation to become ‘‘readily releas- Ca2ϩ-triggered exocytosis, and applied several methods to dissect able’’—that is, competent to fuse rapidly with the plasma mem- out the role of CPXs in the stages of regulated exocytosis from brane in response to a Ca2ϩ trigger. It is generally accepted that vesicle docking to fusion pore formation. Our data demonstrate exocytosis is mediated by SNARE proteins, whose function is fine that CPX II has a positive role in fast vesicle exocytosis by regulating tuned through interaction with other proteins (1). Three SNARE the size of the primed vesicle pools. proteins comprise the minimal machinery underlying vesicle fusion: one vesicle membrane protein synaptobrevin (also called VAMP: Results vesicle-associated membrane protein) and the two plasma mem- CPX Isoform Expression. Chromaffin cells secrete catecholamine brane proteins, syntaxin and SNAP-25 (2–4). These 3 proteins form through the fusion of large dense core vesicles (LDCVs) with the a stable 4-helical bundle called the SNARE core complex that plasma membrane, employing the same conserved SNARE-based drives intracellular membrane fusion. Although in vitro studies have fusion machinery that also mediates synaptic transmitter release shown that these 3 SNARE proteins alone suffice to fuse the membranes of artificial liposomes, the rate of such fusions is orders of magnitude slower than fusion in regulated exocytosis, which Author contributions: H.C., J.B.S., N.B., and R.H.C. designed research; H.C., K.R., F.V., S.T., takes place on a millisecond time scale (5, 6), indicating that K.H., and J.B.S. performed research; K.R. and S.T. contributed new reagents/analytic tools; H.C., K.R., F.V., K.H., and J.B.S. analyzed data; and H.C., J.B.S., N.B., and R.H.C. wrote the additional factors are involved in fast regulated secretion. paper. Complexin (CPX) is a small neuronal protein that binds very The authors declare no conflict of interest. rapidly (Ϸ5 ϫ 107MϪ1⅐sϪ1) and with a 1:1 stoichiometry and high 1Present address: Division of Biology 216-76, California Institute of Technology, Pasadena, affinity to the SNARE core complex (7–12), which is indicative of CA 91125. a regulatory role in fast exocytosis. Four CPX isoforms have been 2Present address: Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol identified in vertebrates, CPXI-IV(13). They all are enriched University, Bangkok 10700, Thailand. particularly in the nervous system, where they exhibit a differential 3Present address: Rudolf-Boehm-Institut fu¨r Pharmakologie und Toxikologie, Universita¨t distribution, but some CPXs are also found in other tissues (7, 13). Leipzig, Ha¨rtelstrasse 16-18, 04107 Leipzig, Germany. The function of CPXs in regulated exocytosis is still a matter of 4To whom correspondence should be addressed. E-mail: [email protected]. controversy. Several early studies indicated that CPXs play a This article contains supporting information online at www.pnas.org/cgi/content/full/ negative role in regulated exocytosis. Injection of recombinant CPX 0810232105/DCSupplemental. II caused depression of release, presynaptic injection of anti-CPX © 2008 by The National Academy of Sciences of the USA 19538–19543 ͉ PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810232105 Downloaded by guest on September 25, 2021 SNARE proteins are known to play a role in the behavior of the fusion pore (27–30)—the aqueous pore that initially connects the vesicle lumen and extracellular space during exocytosis—we tested whether the kinetics of fusion pore dilation is altered in the CPX IIϪ/Ϫ chromaffin cells. We used amperometry, an electrochemical technique that directly reports the kinetics and amount of cate- cholamine release from individual vesicles. Our results show that, for all of the parameters examined, there is no robust difference between CPX IIϪ/Ϫ and CPX IIϩ/ϩ mouse chromaffin cells (Fig. 2), indicating that CPX does not affect the fusion pore formation and dilation step of Ca2ϩ-triggered exocytosis, or its effect is very subtle. Secretion Triggered by Trains of Depolarizations. To examine whether other secretory steps are compromised in CPX IIϪ/Ϫ chromaffin cells, we next applied the method of membrane capac- itance measurement, an approach for monitoring changes in the membrane surface area of the entire plasma membrane at milli- second time resolution. We applied a train of 8 100-ms depolar- izations separated by 100-ms intervals (Fig. 3A), and recorded the capacitance increase (⌬Cm) (Fig. 3C), to obtain a measure of the secretion capacity and dynamics. Although the total Ca2ϩ influx was the same for CPX IIϪ/Ϫ and CPX IIϩ/ϩ cells (Fig. 3 B and F), the total capacitance increase was significantly smaller in CPX IIϪ/Ϫ cells (Fig. 3 C and G). The most significant differences in ⌬Cm occurred during the earlier depolarizations, whereas the ⌬Cm in response to later depolarizations, especially to the 7th and 8th depolarizations, were indistinguishable (Fig. 3E). The selective reduction in the response to the earlier depolarizations of the train is consistent with a function of CPX II in rendering vesicles release-ready, thereby increasing the size of the releasable vesicle pools. Secretion Triggered by Flash Photolysis of Caged Ca2؉. The most direct approach to measure the size of the functional vesicle pools Fig. 1. CPXs expression. (A) Western blot analyses of CPX I and CPX II in chromaffin cells is to monitor the ⌬Cm in response to a uniform expression levels in homogenates of brain (5 ␮g per lane) and adrenal gland and global step elevation of the cytosolic Ca2ϩ concentration (50 ␮g per lane) from mice of the indicated CPX I and CPX II genotypes. (B) 2ϩ 2ϩ ([Ca ]i), achieved by photolysis of caged Ca compounds. The (Left) Western blot analyses of CPX expression levels in homogenates of brain multiphasic rising C trace can be fitted with a triple exponential (5 ␮g per lane) and adrenal gland (50 ␮g per lane) from mice of the indicated m CPX II genotypes.
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