omega-Conotoxins block neurotransmission in the rat vas deferens by binding to different presynaptic sites on the N-type Ca2+ channel

Electrically-induced twitch responses of the prostatic segment of vas deferens (0.1 Hz, 65 V, 1 ms) are mainly due to the transient presynaptic release of ATP, which acts postsynaptically on non-adrenergic receptors to contract smooth muscle cells. These responses were fully blocked by nanomolar concentrations of the omega-conotoxins GVIA, MVIIA, and MVIIC, most likely by inhibiting Ca2+ entry through presynaptic N-type Ca2+ channels controlling the release of ATP. Repeated washout of the toxins allowed the recovery of contractions, except for omega-conotoxin GVIA, whose inhibitory effects remained unchanged for at least 60 min. In addition, micromolar concentrations of omega-conotoxin MVIIC were unable to protect against the irreversible inhibition of twitch contractions induced by nanomolar concentrations of omega-conotoxin GVIA, At low extracellular Ca2+ concentrations (1.5 mM), 20 nM of omega-conotoxin GVIA or MVIIA inhibited completely the twitch contractions in about 10 min. In 5 mM Ca2+ the blockade of twitch contractions after 10 min was 70% for both toxins. In 1.5 mM Ca2+ omega-conotoxin MVIIC (1 mu M) inhibited completely the twitch contraction after 10 min. In 5 mM Ca2+ blockade developed very slowly and was very poor after 30 min, omega-conotoxin MVIIC depressed the response by only 20%. These results are compatible with the idea that the three omega-conotoxins block the purinergic neurotransmission of the vas deferens by acting on presynaptic N-type voltage-dependent Ca2+ channels. However, omega-conotoxin MVIIC seems to bind to sites different from those recognised by omega-conotoxin GVIA and MVIIA, which are markedly differentiated by their Ca2+ requirements for binding to their receptors.