Effects of calciseptine on unitary barium channel currents in guinea-pig portal vein

Abstract

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 μM) 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.