Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction.

1. The effects of the voltage-dependent calcium channel (VDCC) blockers omega-agatoxin IVA (omega-AgaIVA), omega-conotoxin GVIA (omega-CgTx), omega-conotoxin MVIIC (omega-MVIIC) and omega-conotoxin MVIID (omega-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of omega-AgaIVA and omega-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker omega-AgaIVA (100 nM) and P- Q- and N-type channel blockers omega-MVIIC (1 microM) and omega-MVIID (3 microM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker omega-CgTx (5 microM) was ineffective. 3. The process of release was much more sensitive to omega-MVIIC (IC50 = 39 nM) than to omega-MVIID (IC50 = 1.4 microM). After almost completely blocking transmitter release (quantal content approximately 0.3% of its control value) with 3 microM omega-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of approximately 20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 +/- 0.04 to 4.8 +/- 1.4). 4. Nerve-evoked transmitter release in a low Ca(2+)-high Mg2+ medium (low release probability, quantal content = 2 +/- 0.1) had the same sensitivity to omega-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K(+)-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of omega-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33 degrees C. Conversely, the effect of omega-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10-30 nM) of omega-AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 microM omega-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with omega-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.