A Rho-related GTPase Is Involved in Ca2+-dependent Neurotransmitter Exocytosis

doi:10.1074/jbc.275.11.7764

A Rho-related GTPase Is Involved in Ca²⁺-dependent Neurotransmitter Exocytosis*

From the ^‡Laboratoire de Neurobiologie Cellulaire, CNRS, UPR 9009 and ^¶INSERM, U-338 Biologie de la Communication Cellulaire, F-67084 Strasbourg Cédex,^**Toxines Microbiennes, Institut Pasteur, F-75724 Paris Cédex 15, and ^‡INSERM, U-452 Biologie Cellulaire et Moléculaire des Microorganismes et de leurs Toxines, F-06107 Nice Cédex 2, France

Abstract

Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca²⁺-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca²⁺-dependent neuroexocytosis.

Footnotes

↵* This work was supported by grants from the Association Française contre les Myopathies and the Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires and by Direction des Systèmes des Forces et de la Prospective Contract 99-34-038 (to B. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: Dept. of Neurobiology, Duke University Medical Center, Durham, 27710 NC.
↵‖ Present address: Dipt. di Farmacologia e Fisiologia Umana, Facoltà di Medicina e Chirurgia, Università di Bari, I-70124 Bari, Italy.
↵§§ To whom correspondence should be addressed: Lab. de Neurobiologie Cellulaire, CNRS, 5 rue Blaise Pascal, F-67084 Strasbourg Cédex, France. Tel.: 33-3-88-45-66-77; Fax: 33-3-88-60-16-64; E-mail: poulain@neurochem.u-strasbg.fr.
Abbreviations:

ACh

acetylcholine

LT

lethal toxin

CNF1

cytotoxic necrotizing factor 1

ToxB

toxin B

PPF

paired-pulse facilitation
- Received October 14, 1999.
- Revision received December 8, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.