Rab3a deletion reduces vesicle docking and transmitter release at the mouse diaphragm synapse.
Rab3a is a small GTP binding protein associated with presynaptic vesicles that is thought to regulate vesicle targeting to active zones. Although this rab3a function implies that vesicle docking and action potential-evoked release might be inhibited in rab3a gene-deleted synapses, such inhibition has never been demonstrated. To investigate vesicle docking at the neuromuscular junction of rab3a gene-deleted (rab3a(-)) mice, we performed electron microscopy analysis of the diaphragm slow-fatigue (type I) synapses. We found a significant (26%) reduction in the number of vesicles docked to the presynaptic membrane in rab3a(-) terminals, although intraterminal vesicles were not affected. Aiming to detect possible changes in quantal release due to rab3a gene deletion, we minimized the variability between preparations employing focal recordings of synaptic responses from visualized type I endplates. We found a significant decrease in both evoked (27% reduction in quantal content) and spontaneous (28% reduction in mini frequency) quantal release. The decrease in the evoked release produced by rab3a deletion was most pronounced at reduced extracellular Ca(2+) concentrations (over 50% decrease at 0.5 and 0.2 mM Ca(2+)). By manipulating extracellular calcium, we demonstrated that calcium cooperativity is not altered in rab3a(-) synapses, however calcium sensitivity of quantal release is affected. Thus, we demonstrated that rab3a positively regulates docking and basal quantal release at the mouse neuromuscular junction. This result is consistent with the proposed role of rab3a in trafficking and targeting vesicles to the active zones.
Coleman
W L
WL
Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA.
Bill
C A
CA
Bykhovskaia
M
M
D000818:Animals; D002118:Calcium / Q000172:deficiency; D020013:Calcium Signaling / Q000235:genetics; D003964:Diaphragm / Q000294:innervation* / Q000503:physiopathology; D019706:Excitatory Postsynaptic Potentials / Q000235:genetics; D005089:Exocytosis / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D009046:Motor Neurons / Q000378:metabolism / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000648:ultrastructure; D020511:Neuromuscular Junction Diseases / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000235:genetics*; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D020826:rab3A GTP-Binding Protein / Q000235:genetics*
D018377:Neurotransmitter Agents; D020826:rab3A GTP-Binding Protein; D002118:Calcium