The effect of α-latrotoxin on the neurosecretory PC12 cell line: Studies on toxin binding and stimulation of transmitter release

α-Latrotoxin of black widow spider venom was found to bind with high affinity (K_A = 1.8·10⁹M^-1) to specific sites present in discrete number (∼6300/cell, ∼12/aμn2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (∼ 2-fold) in the presence of mM concentrations of various divalent cations (Ca²⁺, Mn²⁺ and Co²⁺) while Ba²⁺ and Sr²⁺ had a smaller effect and Mg²⁺ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The α-latrotoxin-induced stimulation of ³H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca²⁺-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca²⁺-free, Mg²⁺-containing Ringer. Introduction of Ca²⁺ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na²⁺ and Ca²⁺ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca²⁺ and Mg²⁺ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and l.5mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca²⁺ and Mg²⁺, the relative efficacy being Sr²⁺ > Ca²⁺ ≥> Ba²⁺ > Mn²⁺ > Mg²⁺ > Co²⁺. Moreover, the response occurring at suboptimal concentration of Ca²⁺ (0.4 mM) was potentiated by the concomitant addition of either Mg²⁺, Mn²⁺ or Co²⁺. The effect(s) of divalent cations in supporting the α-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because 1. (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding 2. (b) Ca²⁺ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca²⁺-free medium. Delays in the release responses were observed when cells were exposed to αLTx in Na²⁺-free, glucosamine or methylamine-based media, or depolarized with high K²⁺ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg²⁺ to support the αLTx response was considerably decreased. Taken together with previous data of the literature these results appear consistent with the hypothesis that αLTx acts by activating a 'non-conventional' cation channel of low ionic specificity in the plasma membrane. This would result in a stimulated influx of various divalent cations, not only Ca²⁺ but also Mg²⁺ and the others investigated, which would then be able to stimulate the release response, although with different efficacy.