Rac GTPase plays an essential role in exocytosis by controlling the fusion competence of release sites.
The role of small GTPases of the Rho family in synaptic functions has been addressed by analyzing the effects of lethal toxin (LT) from Clostridium sordellii strain IP82 (LT82) on neurotransmitter release at evoked identified synapses in the buccal ganglion of Aplysia. LT82 is a large monoglucosyltranferase that uses UDP-glucose as cofactor and glucosylates Rac (a small GTPase related to Rho), and Ras, Ral, and Rap (three GTPases of the Ras family). Intraneuronal application of LT (50 nm) rapidly inhibits evoked acetylcholine (ACh) release as monitored electrophysiologically. Injection of the catalytic domain of the toxin similarly blocked ACh release, but not when key amino acids needed for glucosylation were mutated. Intraneuronal application of competitive nucleotide sugars that differentially prevent glucosylation of Rac- and Ras-related GTPases, and the use of a toxin variant that affects a different spectrum of small GTPases, established that glucosylation of Rac is responsible for the reduction in ACh release. To determine the quantal release parameters affected by Rac glucosylation, we developed a nonstationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action. The results indicate that neither the quantal size nor the average probability for release were affected by lethal toxin action. ACh release blockage by LT82 was only caused by a reduction in the number of functional release sites. This reveals that after docking of synaptic vesicles, vesicular Rac stimulates a membrane effector (or effectors) essential for the fusion competence of the exocytotic sites.
Humeau
Yann
Y
Neurotransmission et Sécrétion Neuroendocrine, UPR2356 du Centre National de la Recherche Scientifique, IFR-37 des Neurosciences, F-67084 Strasbourg Cedex, France.
Popoff
Michel R
MR
Kojima
Hiroshi
H
Doussau
Frédéric
F
Poulain
Bernard
B
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D006031:Glycosylation / Q000187:drug effects; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008845:Microinjections; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013569:Synapses / Q000187:drug effects / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*
D001427:Bacterial Toxins; C072292:lethal toxin LT, Clostridium sordellii; D020744:rac GTP-Binding Proteins; D008274:Magnesium; D000109:Acetylcholine; D002118:Calcium