SNARE proteins mediate the release of neurotransmitters by catalyzing the fusion of synaptic vesicles with the plasma membrane. However, the extent that SNAREs function in vesicle fusion remains controversial. In vitro experiments demonstrate that reconstituted SNAREs are sufficient for synaptic vesicles to fuse. In contrast, the elimination of individual SNAREs in vivo does not always eliminate fusion. Another component has been proposed to mediate membrane fusion: the Vo sector of the V-type ATPase proton pump. We are testing the hypothesis that Vo functions in synaptic vesicle fusion by analyzing neurotransmission with electrophysiology and imaging. The V-ATPase consists of two sectors: an ATPase-hydrolyzing V1 sector and a Vo proton-translocating sector. The Vo hypothesis predicts mutations in the subunits that comprise the Vo sector would eliminate vesicle fusion whereas mutations in the V1 sector would not. In C. elegans there are mutations in both sectors.
vha-12(
n2915) disrupts the B subunit of V1 whereas
unc-32(
e189) eliminates a neural-specific isoform of the Vo a subunit. If the Vo hypothesis is true we expect a defect in acidification in both mutants and a synaptic vesicle fusion defect in the Vo mutant
unc-32(
e189). To test whether synaptic vesicles are normally acidified by the V-ATPase, we have targeted a pH-sensitive GFP (synaptopHluorin) to the lumen of synaptic vesicles. SynaptopHluorin fluorescence is quenched at low pH. Consistent with an acidification defect, synaptopHlourin fluorescence in
vha-12(
n2915) and
unc-32(
e189) is increased two-fold from the control strain. At the neuromuscular junction, the fusion of a single synaptic vesicle loaded with transmitter elicits a postsynaptic current in the muscle (mPSCs). We find the frequency of mPSCs is moderately reduced in
vha-12(
n2915), and the frequency is greatly reduced in
unc-32(
e189). Since acidification is a prerequisite for neurotransmitter loading, we were surprised to find that the average mPSC amplitude was normal in both
vha-12(
n2915) and
unc-32(
e189). To distinguish whether the reduction in mPSC rate is due to a defect in fusion or a defect in transmitter loading we are labeling recycling vesicles with FM dyes. A decreased FM dye destaining rate in
unc-32(
e189) will indicate a fusion defect.