Multiple types of Ca2+ channels in mouse motor nerve terminals.

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either omega-agatoxin IVA [omega-Aga-IVA (0.3 microM), a blocker of P-type Ca2+ channels] or omega-conotoxin MVIIC [omega-CTx-MVIIC (2 microM), a P- and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 microM, a blocker of L-type Ca2+ channels) plus omega-conotoxin GVIA [omega-CTx-GVIA (2 microM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3,4-diaminopyridine (3,4-DAP, a K+-channel blocker) or high Ca2+ (10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3,4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both omega-Aga-IVA and omgea-CTx-MVIIC; omega-Aga-IVA exerted only a partial inhibitory effect on endplate potentials, and the omega-Aga-IVA-resistant component was further inhibited by omega-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 microM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by omega-CTx-MVIIC with further incomplete inhibition by omega-Aga-IVA. Cd2+ (300 microM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 microM) with omega-CTx-GVIA (2 microM) was without inhibitory effect. We conclude that multiple types of Ca2+ channels, i.e. omega-Aga-IVA-sensitive, omega-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals.