Syntaxin modulation of calcium channels in cortical synaptosomes as revealed by botulinum toxin C1

When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oo cytes with N- or P/Q-type Ca2+ channels, it promotes their inactivation (Be zprozvanny et al., 1995; Wiser et al., 1996, 1999; Degtiar et al., 2000) (I . B. Bezprozvanny, P. Zhong, R. H. Scheller, and R. W. Tsien, unpublished o bservations). These findings led to the hypothesis that syntaxin influences Ca2+ channel function in presynaptic endings, in a reversal of the convent ional flow of information from Ca2+ channels to the release machinery. We e xamined this effect in isolated mammalian nerve terminals (synaptosomes). B otulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied t o rat neocortical synaptosomes at concentrations that completely blocked ne urotransmitter release. This treatment altered the pattern of Ca2+ entry mo nitored with fura-2. Whereas the initial Ca2+ rise induced by depolarizatio n with K+-rich solution was unchanged, late Ca2+ entry was strongly augment ed by syntaxin cleavage. Similar results were obtained when Ca2+ influx aro se from repetitive firing induced by the K+-channel blocker 4-aminopyridine . Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 wit h BoNtE failed to produce a significant change in Ca2+ entry. The BoNtC1-in duced alteration in Ca2+ signaling was specific to voltage-gated Ca2+ chann els, not Ca2+ extrusion or buffering, and it involved N-, P/Q- and R-type c hannels, the high voltage-activated channels most intimately associated wit h presynaptic release machinery. The modulatory effect of syntaxin was not immediately manifest when synaptosomes had been K+-predepolarized in the ab sence of external Ca2+, but developed with a delay after admission of Ca2+, suggesting that vesicular turnover may be necessary to make syntaxin avail able for its stabilizing effect on Ca2+ channel inactivation.