Abstract
alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.
Highlights
␣-Latrotoxin (LTX)1 causes massive release of neurotransmitters (NTs) and is widely used to study exocytosis of synaptic and large dense-cored vesicles in neuronal and endocrine cells [1,2,3,4]
Tetramers—La3ϩ blocks two types of LTX-evoked amino acid (AA) release: Ca2ϩ-independent and delayed Ca2ϩ-dependent. These release types require the presence of LTX pores [19]. These findings suggest that La3ϩ inhibits AA release by affecting LTX pores
We found that 150 mM sucrose stimulated Ca2ϩ-independent [14C]glutamic acid (Glu) release from synaptosomes (ϳ9% of total AA content; e.g. Fig. 7C, control); this secretion was largely vesicular because pretreatment of terminals with bafilomycin A1 (BAF) decreased it by 65 Ϯ15%
Summary
Materials—␣-Latrotoxin was purified from spider venom [19]. Pure BoNT/E was a kind gift of Prof. During the post-loading incubation, some synaptosomes were treated with: 400 nM BoNT/E (2 h, eliminating 90% of SNAP-25) [20], 100 M BAPTA-AM (1 h) [20], 1 M BAF (1 h) [22], 10 M U73122 (15 min), 10 M TG (30 min, no less TG is required to cause specific effects in brain preparations) [23, 24], or 3 M phenylarsine oxide (PAO, 15 min) [21] The latter three drugs were included in subsequent buffers. A and B, optimal time for loading terminals with radiolabeled AAs. Rat synaptosomes were incubated with [14C]Glu at 37 °C for 5 min, washed, and kept at 37 °C for indicated times prior to a final wash and a 1-min stimulation with 25 mM Kϩ Ϯ 2.5 mM Ca2ϩ. Statistical significance of differences between control and test values (probability that the values are equal) was assessed by paired Student’s t test (p values are shown near the respective points; NS, nonsignificant) or by the Kolmogorov-Smirnov twosample test
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