Effects of synthesized calciseptine (CaS), found naturally in the venom of the black mamba, on voltage-dependent Ca2+ channels in smooth muscle cells of the guinea-pig portal vein were investigated. In the whole-cell voltage-clamp configuration, extracellular application of CaS (>/= 10 nM) inhibited the inward current in a concentration- and voltage-dependent manner at a holding potential of -90 mV. The Ca2+ current recorded at a high holding potential (-50 mV) was approximately 8 times more sensitive to CaS than that at a more negative holding potential (-90 mV). CaS (50 nM) shifted to the left the steady-state inactivation curve obtained by using single 8-s conditioning pulses of various amplitudes. When CaS (>/= 200 nM) was present in the pipette, the Ca2+ current remained for the duration of the experiments (more than 60 min) in the whole-cell configuration. Two different Ca2+ channel conductances are present in this tissue (25-pS and 12-pS channels). Both channels are blocked by dihydropyridine (DHP) derivatives, but have different sensitivities. In the cell-attached condition, CaS hardly changed the activity of either unitary Ca2+ channel current. To prevent the "run down" of the Ca2+ channels in cell-free conditions, we added cardiac cytosol, a supernatant from homogenized cardiac cells and an endogenous Ca2+ channel activating factor, in the pipette. The unitary Ca2+ channel currents were then recorded using the outside-out membrane patch configuration. Application of CaS (1 microM) in the bath completely blocked the open events of the 25-pS Ca2+ channel. CaS (10 nM) in the bath reduced the mean open time and channel availability, resulting in a decrease in the open probability of the 25-pS channel currents without affecting the amplitude of the single-channel conductance. CaS also reduced the open probability (though less potently) and channel availability of the 12-pS Ca2+ channel without a change in its amplitude. From these results, we conclude that CaS has inhibitory effects on the voltage-dependent Ca2+ current that are similar to those of DHP derivatives and that it acts from the outside of the membrane.