α-Latrotoxin and related toxins

alpha-Latrotoxin (alpha-LTX) from black widow spider venom induces exhaustive release of neurotransmitters from vertebrate nerve terminals and endocrine cells. This 130-kDa protein has been employed for many years as a molecular tool to study exocytosis. However, its action is complex: in neurons, alpha-LTX induces massive secretion both in the presence of extracellular Ca(2+) (Ca(2+) (e)) and in its absence; in endocrine cells, it usually requires Ca(2+) (e). To use this toxin for further dissection of secretory mechanisms, one needs an in-depth understanding of its functions. One such function that explains some alpha-LTX effects is its ability to form cation-permeable channels in artificial lipid bilayers. The mechanism of alpha-LTX pore formation, revealed by cryo-electron microscopy, involves toxin assembly into homotetrameric complexes which harbor a central channel and can insert into lipid membranes. However, in biological membranes, alpha-LTX cannot exert its actions without binding to specific receptors of the plasma membrane. Three proteins with distinct structures have been found to bind alpha-LTX: neurexin Ialpha, latrophilin 1, and receptor-like protein tyrosine phosphatase sigma. Upon binding a receptor, alpha-LTX forms channels permeable to cations and small molecules; the toxin may also activate the receptor. To distinguish between the pore- and receptor-mediated effects, and to study structure-function relationships in the toxin, alpha-LTX mutants have been used.

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α-Latrotoxin and Its Receptors

A novel fluorescent Na+ indicator, Na(+)-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na+ concentration ([Na+]i) of synaptosomes. The dye, when loaded into synaptosomes in the form of its acetoxymethyl ester, was responsive to changes of [Na+]. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na+ concentration was equilibrated with different concentrations of extracellular Na+ in the presence of 2 microM gramicidin D. The basal value of [Na+]i in synaptosomes in the presence of 140 mM extracellular Na+ was found to be 10.9 +/- 1.8 mM. Veratridine, which opens potential-dependent Na+ channels, caused a sudden increase in [Na+]i in a concentration-dependent manner (1-20 microM), whereas the effect of ouabain (20 and 50 microM), the inhibitor of the plasma membrane Na+,K(+)-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 microM tetrodotoxin. alpha-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19-1.5 nM). This report confirms our earlier finding demonstrating a Na(+)-dependent component in the action of alpha-latrotoxin, and shows that changes in [Na+]i in synaptosomes can be followed by SBFI.

Detection of intracellular free Na+ concentration of synaptosomes by a fluorescent indicator, Na(+)-binding benzofuran isophthalate: the effect of veratridine, ouabain, and alpha-latrotoxin.

Calcium ions play many important roles in the function of nerve cells. Calcium’s involvement in neurotransmitter release is one of its key roles, and was one of the first to be recognized. Early observations demonstrated that extracellular Ca was required for neurotransmitter release (Harvey and Macintosh 1940). Subsequent studies established that transmitter release was evoked by Ca entry and a rise in [Ca2+]i, the intracellular free Ca concentration. This sequence of events accounted for the external Ca dependence (Katz 1969; Baker 1972; Llinas et al. 1981; Drapeau and Blaustein 1983 a, b). Moreover, the modulation of synaptic transmission, and related fundamental aspects of transmitter release associated with memory and learning, are all profoundly influenced by Ca.

Calcium and Synaptic Function

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5-min delay following toxin addition. Maitotoxin-induced 45Ca2+ influxes could not be blocked by either 1 μM verapamil, 1 μM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50= 41 μM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+ the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 μM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose-response curves for H+ ion inhibition of maitotoxin-induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3- and HCO3-/Cl- ions.



It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore-forming or channel-forming activity of a non-classical type. Some properties of maitotoxin resemble those of α-latrotoxin, others those of pore-forming agents such as melittin or α-toxin of Staphylococcus aureus.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb14149.x

New insights into maitotoxin action

α-Latrotoxin, a polypeptide and potent presynaptic neurotoxin, interacts with artificial lipid bilayers inducing a large increase of conductance, when added to one ( cis side) of the two bathing solutions. These conductance changes are due to the presence of channels which, in 0.1 m monovalent cation solution, have conductances between 100 and 400 pS. Current-voltage relations of macroscopic and single channel conductances show marked non-ohmic behaviour being reduced at positive potentials, referred to cis side as virtual ground. The decrease in conductance at high positive voltage is modulated by lipid composition and abolished by digestion with pronase of a trans -bilayer protruding domain of the protein. The results are consistent with the notion that α-latrotoxin forms channels and provide evidence that the molecule is endowed with a specific mode of insertion and orientation in lipid bilayers.

R. Rolandi

Papers

Trans-Bilayer orientation and voltage-dependence of α-latrotoxin-induced channels