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

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Abstract
The effects of the voltage‐dependent calcium channel (VDCC) blockers ω‐agatoxin IVA (ω‐AgaIVA), ω‐conotoxin GVIA (ω‐CgTx), ω‐conotoxin MVIIC (ω‐MVIIC) and ω‐conotoxin MVIID (ω‐MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of ω‐AgaIVA and ω‐MVIIC were also evaluated on the perineurial calcium and calcium‐dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. The P‐ and Q‐type VDCC blocker ω‐AgaIVA (100 nM) and P‐ Q‐ and N‐type channel blockers ω‐MVIIC (1 μM) and ω‐MVIID (3 μM) strongly reduced transmitter release (>80–90% blockade) whereas the selective N‐type channel blocker ω‐CgTx (5 μM) was ineffective. The process of release was much more sensitive to ω‐MVIIC (IC50=39 nM) than to ω‐MVIID (IC50=1.4 μM). After almost completely blocking transmitter release (quantal content ∼0.3% of its control value) with 3 μMω‐MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of ∼20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2±0.04 to 4.8±1.4). Nerve‐evoked transmitter release in a low Ca2+‐high Mg2+ medium (low release probability, quantal content = 2±0.1) had the same sensitivity to ω‐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 ω‐AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31–33°C. Conversely, the effect of ω‐AgaIVA persisted even after two hours of toxin washout at room temperature. Both the calcium and calcium‐dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10–30 nM) of ω‐AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 μMω‐MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with ω‐MVIIC. 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. British Journal of Pharmacology (1997) 121, 1531–1540; doi:10.1038/sj.bjp.0701290