Molecular mechanisms of COMPLEXIN fusion clamp function in synaptic exocytosis revealed in a new Drosophila mutant.

The COMPLEXIN (CPX) proteins play a critical role in synaptic vesicle fusion and neurotransmitter release. Previous studies demonstrated that CPX functions in both activation of evoked neurotransmitter release and inhibition/clamping of spontaneous synaptic vesicle fusion. Here we report a new cpx mutant in Drosophila melanogaster, cpx(1257), revealing spatially defined and separable pools of CPX which make distinct contributions to the activation and clamping functions. In cpx(1257), lack of only the last C-terminal amino acid of CPX is predicted to disrupt prenylation and membrane targeting of CPX. Immunocytochemical analysis established localization of wild-type CPX to active zone (AZ) regions containing neurotransmitter release sites as well as broader presynaptic membrane compartments including synaptic vesicles. Parallel biochemical studies confirmed CPX membrane association and demonstrated robust binding interactions of CPX with all three SNAREs. This is in contrast to the cpx(1257) mutant, in which AZ localization of CPX persists but general membrane localization and, surprisingly, the bulk of CPX-SNARE protein interactions are abolished. Furthermore, electrophysiological analysis of neuromuscular synapses revealed interesting differences between cpx(1257) and a cpx null mutant. The cpx null exhibited a marked decrease in the EPSC amplitude, slowed EPSC rise and decay times and an increased mEPSC frequency with respect to wild-type. In contrast, cpx(1257) exhibited a wild-type EPSC with an increased mEPSC frequency and thus a selective failure to clamp spontaneous release. These results indicate that spatially distinct and separable interactions of CPX with presynaptic membranes and SNARE proteins mediate separable activation and clamping functions of CPX in neurotransmitter release.

Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission.

Mitochondria located within neuronal presynaptic terminals have been shown to play important roles in the release of chemical neurotransmitters. In the present study, a genetic screen for synaptic transmission mutants of Drosophila has identified the first mutation in a Drosophila homolog of the mitochondrial protein P32. Although P32 is highly conserved and has been studied extensively, its physiological role in mitochondria remains unknown and it has not previously been implicated in neural function. The Drosophila P32 mutant, referred to as dp32(EC1), exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in the amplitude of excitatory postsynaptic currents (EPSC) and indicated that dP32 functions in neurotransmitter release. These studies are the first to address P32 function in Drosophila and expand our knowledge of mitochondrial proteins contributing to synaptic transmission.