Nerve-evoked synchronous release and high K+ -induced quantal events are regulated separately by synaptotagmin I at Drosophila neuromuscular junctions.

The distal Ca2+- binding domain of synaptotagmin I ( Syt I), C2B, has two Ca2+- binding sites. To study their function in Drosophila, pairs of aspartates were mutated to asparagines and the mutated syt I was expressed in the syt I - null background ( P[syt I-B-D1,I-2N] and P[syt I-B-D3,I-4N]). We examined the effects of these mutations on nerve-evoked synchronous synaptic transmission and high K+- induced quantal events at embryonic neuromuscular junctions. The P[sytI(B-D1,2N)] mutation virtually abolished synaptic transmission, whereas the P[ syt I-B-D3,I-4N] mutation strongly reduced but did not abolish it. The quantal content in P[ syt IB-D3,4N] increased with the external Ca2+ concentration, [ Ca2+](e), with a slope of 1.86 in double-logarithmic plot, whereas that of control was 2.88. In high K+ solutions the quantal event frequency in P[ syt I-B-D3,I-4N] increased progressively with [ Ca2+](e) between 0 and 0.15 mM as in control. In contrast, in P[ syt I-B-D1,I-2N] the event frequency did not increase progressively between 0 and 0.15 mM and was significantly lower at 0.15 than at 0.05 mM [ Ca2+](e). The P[ syt I-B-D1,I-2N] mutation inhibits high K+- induced quantal release in a narrow range of [ Ca2(+)](e) ( negative regulatory function). When Sr2+ substituted for Ca2+, nerve-evoked synchronous synaptic transmission was severely depressed and delayed asynchronous release was appreciably increased in control embryos. In high K+ solutions with Sr2+, the quantal event frequency was higher than that in Ca2+ and increased progressively with [ Sr2+](e) in control and in both mutants. Sr2+ partially substitutes for Ca2+ in synchronous release but does not support the negative regulatory function of Syt I.