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

When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oocytes with N- or P/Q-type Ca2+ channels, it promotes their inactivation (Bezprozvanny 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1995; Wiser et al., 1996, 1999; Degtiar et al., 2000) (I. B. Bezprozvanny, P. Zhong, R. H. Scheller, and R. W. Tsien, unpublished observations). These findings led to the hypothesis that syntaxin influences Ca2+ channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca2+ channels to the release machinery. We examined this effect in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely blocked neurotransmitter release. This treatment altered the pattern of Ca2+ entry monitored with fura-2. Whereas the initial Ca2+ rise induced by depolarization with K+-rich solution was unchanged, late Ca2+ entry was strongly augmented by syntaxin cleavage. Similar results were obtained when Ca2+ influx arose from repetitive firing induced by the K+-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca2+ entry. The BoNtC1-induced alteration in Ca2+ signaling was specific to voltage-gated Ca2+ channels, not Ca2+ extrusion or buffering, and it involved N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin was not immediately manifest when synaptosomes had been K+-predepolarized in the absence of external Ca2+, but developed with a delay after admission of Ca2+, suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca2+ channel inactivation.