title: Nerve-evoked synchronous release and high K+ -induced quantal events are regulated separately by synaptotagmin I at Drosophila neuromuscular junctions.
abstract: The distal Ca(2+)-binding domain of synaptotagmin I (Syt I), C2B, has two Ca(2+)-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 Ca(2+) concentration, [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](e). The P[syt I(B-D1,2N)] mutation inhibits high K(+)-induced quantal release in a narrow range of [Ca(2+)](e) (negative regulatory function). When Sr(2+) substituted for Ca(2+), nerve-evoked synchronous synaptic transmission was severely depressed and delayed asynchronous release was appreciably increased in control embryos. In high K(+) solutions with Sr(2+), the quantal event frequency was higher than that in Ca(2+) and increased progressively with [Sr(2+)](e) in control and in both mutants. Sr(2+) partially substitutes for Ca(2+) in synchronous release but does not support the negative regulatory function of Syt I.
authors.affiliation: Institute for Molecular and Cellular Neuroscience, Gunma University, Maebashi, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Tamura
authors.forename: Jiamei
authors.initials: J
authors.lastname: Hou
authors.forename: Noreen E
authors.initials: NE
authors.lastname: Reist
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kidokoro
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000235:genetics; D004330:Drosophila; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010525:Peripheral Nerves / Q000187:drug effects / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D013324:Strontium / Q000378:metabolism / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects / Q000235:genetics*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050863:Synaptotagmin I; D001224:Aspartic Acid; D011188:Potassium; D002118:Calcium; D013324:Strontium