Penelope's web: using alpha-latrotoxin to untangle the mysteries of exocytosis

Abstract

For more than three decades, the venom of the black widow spider and its principal active components, latrotoxins, have been used to induce release of neurotransmitters and hormones and to study the mechanisms of exocytosis. Given the complex nature of alpha--latrotoxin (alpha-LTX) actions, this research has been continuously overshadowed by many enigmas, misconceptions and perpetual changes of the underlying hypotheses. Some of the toxin's mechanisms of action are still not completely understood. Despite all these difficulties, the extensive work of several generations of neurobiologists has brought about a great deal of fascinating insights into pre-synaptic processes and has led to the discovery of several novel proteins and synaptic systems. For example, alpha-LTX studies have contributed to the widespread acceptance of the vesicular theory of transmitter release. Pre-synaptic receptors for alpha-LTX--neurexins, latrophilins and protein tyrosine phosphatase sigma--and their endogenous ligands have now become centrepieces of their own areas of research, with a potential of uncovering new mechanisms of synapse formation and regulation that may have medical implications. However, any future success of alpha-LTX research will require a better understanding of this unusual natural tool and a more precise dissection of its multiple mechanisms.

Fig. 1

Dissection of the two distinct mechanisms of α-LTX action in the presence and absence of 2 mM Ca²⁺_e. (a) A representative recording at the mouse NMJ of mepps induced by 1 nM wild-type α-LTX in the absence of Ca²⁺_e. (b-d) Similar recordings using wild-type α-LTX, with Ca²⁺_e added at different times (arrowheads). Two phases are clearly visible: clonic (C) and tonic (T). Only the tonic phase occurs in the absence of Ca²⁺; it is characterized by a smooth, slowly rising and falling pattern (dotted lines) and always results in the cessation of spontaneous release events, even after the subsequent addition of Ca²⁺. Clonic exocytosis only occurs in the presence of Ca²⁺ and is additive with tonic release. (e) In a similar recording, on addition of Ca²⁺ (arrowhead), 1 nM LTX^N4C only triggers the clonic phase. This burst-like spontaneous exocytosis continued for 5 hr. (From (Lelianova et al. 2009); modified).

Fig. 2

Generalized scheme of the α-LTX receptors and some of its mechanisms of action at the synapse. The wild-type toxin is dimeric, but it assembles into tetramers in the presence of millimolar Mg²⁺. After binding to its receptors, tetrameric α-LTX inserts itself into the plasma membrane and forms non-selective cation channels. The subsequent influx of Ca²⁺ is able to mediate Ca²⁺-dependent α-LTX evoked neurotransmitter release. Mutant LTX^N4C (dimeric only) is able to stimulate receptor-mediated neurotransmitter release which requires both intracellular and extracellular Ca²⁺. This mode of action may involve voltage-dependent Ca²⁺ channels and is probably mediated by latrophilin 1, which, upon binding the toxin, signals via the PLC cascade. Both neurexin and latrophilin 1 appear to organize VDCCs. Abbreviations used here are: DAG, diacyl glycerol; LPH1, latrophilin 1; LTX 4x, α-LTX tetramers; LTX 2x, α-LTX dimers; NLG, neuroligin; NRX, neurexin; NT, neurotransmitter; PKC, protein kinase C; PreS, presynaptic scaffolding; PostS, postsynaptic scaffolding; SV, synaptic vesicles; VDCC; voltage-dependent Ca²⁺ channels.

Fig. 3

A web of intrigue. A summary of processes (rectangles) and molecules (rounded rectangles) revealed using BWSV and α-LTX. Key proteins, concepts and actions are shown in bold. Solid black lines indicate causal links between findings; solid gray lines show interactions of unknown significance; dotted arrows signify contributions to the understanding of respective phenomena. Other toxins from BWSV, α-ε-latroinsectotoxins (α-ε-LITs) and α-LCT, have largely confirmed the findings made using α-LTX (Rohou et al. 2007).