Nerve-Evoked Synchronous Release and High K+-Induced Quantal Events Are Regulated Separately by Synaptotagmin I at Drosophila Neuromuscular Junctions

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Abstract
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-D1,2N] and P[ syt I B-D3,4N]). 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[ syt I B-D1,2N] mutation virtually abolished synaptic transmission, whereas the P[ syt I B-D3,4N] mutation strongly reduced but did not abolish it. The quantal content in P[ syt I B-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-D3,4N] increased progressively with [Ca2+]e between 0 and 0.15 mM as in control. In contrast, in P[ syt I B-D1,2N] 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-D1,2N] 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.