α-Latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (i) receptor binding/stimulation and (ii) membrane pore formation. Here, we use the toxin mutant, LTXN4C, produced by the insertion of four amino acids into the native sequence, to selectively study the receptor-mediated actions of LTX.
LTXN4C bound to both LTX receptors (latrophilin 1 and neurexin Iα) and greatly increased the frequency of mEPSCs recorded in whole-cell voltage-clamp mode from CA3 pyramidal neurons in rat hippocampal slice cultures (23.5 ± 5-fold, n = 18, P < 0.001 (data shown as means ± S.E.M., paired t test, considered significant if P < 0.05)). LTXN4C acted only via the receptor-mediated mechanism because it failed to form tetramers and ionic pores and its effect was reversible and was not inhibited by La3+ which, in agreement with previous observations (Ashton et al. 2001), perturbed LTX pores.
The action of LTXN4C on mEPSCs was attenuated by the removal of extracellular Ca2+ and by the inhibition of phospholipase C with U73122 (4.2 ± 1.3-fold, n = 5, P < 0.02 and 5 ± 1.5-fold, n = 4, P < 0.05, respectively). Furthermore, both thapsigargin, which depletes Ca2+ stores, and 2-aminoethoxydiphenyl borate, which blocks IP3-induced release of Ca2+ from such stores, essentially abolished the LTXN4C-evoked increase in mEPSC frequency (2 ± 0.5-fold, n = 5, P > 0.7 and 2.1 ± 0.5-fold, n = 9, P > 0.08, respectively).
Measurements using a fluorescent Ca2+ indicator directly demonstrated that LTXN4C increased the basal fluorescence at axonal varicosities (n = 6, P < 0.03); this rise of cytosolic Ca2+ was prevented by thapsigargin (n = 3, P > 0.5), suggesting, together with electrophysiological data, that the receptor-mediated action of LTXN4C involved the mobilization of Ca2+ from intracellular stores.
Finally, in contrast to the wild-type LTX, which inhibited evoked synaptic transmission probably due to pore formation, LTXN4C actually enhanced synaptic currents elicited by electrical stimulation of afferent fibres (1.4 ± 0.1-fold, n = 5, P < 0.04).
We suggest that the mutant LTXN4C, lacking the ionophore-like activity of the wild-type toxin, activates a presynaptic receptor, probably the G protein-coupled latrophilin 1, and stimulates phospholipase C, leading to subsequent Ca2+ release from intracellular stores and the enhancement of synaptic vesicle exocytosis.
This work was supported by the MRC and Wellcome Trust.