Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I

doi:10.1073/pnas.201398798

Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I

^*Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; ^§Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, Hermann-Rein Strasse 3, D-37005 Gottingen, Germany; and ^‡‡Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235

Contributed by Erwin Neher

Abstract

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca²⁺-dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca²⁺-uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca²⁺-dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca²⁺-sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.

Footnotes

↵ † To whom reprint requests should be sent at the present address: Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg Onderwijs & Navorsing, B-3000 Leuven, Belgium. E-mail: Thomas.Voets{at}med.kuleuven.ac.be.
↵ ‡ Present address: Department of Otolaryngology, Göttingen University Medical School, 37073 Göttingen, Germany.
↵ ¶ Present address: Department of Physiology, Uppsala University Biomedical Center, Box 752, 75123 Uppsala, Sweden.
↵ ‖ Present address: Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, 1333 San Pablo Street, Los Angeles, CA 90089-9142.
↵ ** Present address: Department of Comparative Genetics, SmithKline Beecham, Third Avenue, Harlow, Essex CM19 5AW, United Kingdom.
Abbreviations:

LDCV,

large dense-core vesicle;

SNARE,

soluble N-ethylmaleimide-sensitive factor attachment protein receptor;

Cm,

membrane capacitance;

RRP,

readily releasable pool;

SRP,

slowly releasable pool;

[Ca2+]i,

intracellular Ca2+ concentration