A Rho-related GTPase is involved in Ca2+-dependent neurotransmitter exocytosis

Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the acti n cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible impli cation in Ca2+-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA,RhoB, Rac1, and Cdc42 are pre sent in rat brain synaptosomes; however, only Rad was associates with highl y purified synaptic vesicles. To determine the synaptic function of these G TPases, toxins that impair Rho-related proteins were microinjected into Apl ysia neurons. We used lethal toxin from Clostridium sordellii, which inacti vates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium bo;botulinum and cytotoxic nec rotizing 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 Rad, was implicated in the control of neuro transmitter release. Strikingly, blockage of acetylcholine release by letha l toxin and toxin B could be completely removed in <1 s by high frequency s timulation of nerve terminals. Further characterization of the inhibitory a ction produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca2+-dependent neuroexocytosis.