VOLTAGE-DEPENDENT SODIUM AND POTASSIUM, BUT NO CALCIUM CONDUCTANCES IN DDT1 MF-2 SMOOTH-MUSCLE CELLS

Voltage-dependent inward and outward membrane currents were investigated in the DDT1 MF-2 smooth muscle cell line using the whole-cell patch-clamp technique. Application of a pulse protocol with subsequent depolarizing voltage steps elicited an inactivating inward current and a non-inactivating outward current. The outward current was activated at membrane potentials more positive than -35mV, with tau-act = 30-40 ms. The outward current was blocked by tetraethylammonium (NEt4Cl) and 3,4-aminopyridine in a dose-dependent manner (EC50 of 5 mM and 0.5 mM, respectively). The amplitude of the outward current was linked to the potassium equilibrium potential (V(ek)), and tail currents reversed near V(ek). The outward current was completely abolished when intracellular potassium was substituted by 106 mM caesium and 20 mM NEt4Cl. The inward current was activated at potentials more positive than -30 mV with tau-act of 1.6-2.5 ms, and with tau-inact of 1.7-3.0 ms. Steady-state inactivation was 50% at a holding potential of -40 mV. The inward current was blocked by tetrodotoxin (EC50 of 0.15-mu-M) and dependent on the reversal potential for sodium. Voltage-dependent calcium currents could not be detected. Further, the cytoplasmic free calcium concentration, as measured using Indo-1 fluorescence, was not changed during high-potassium(40 mM)-induced depolarization. In contrast, contraction of freshly obtained hamster vas deferens tissue elicited by high-potassium(40 mM)-induced depolarization was largely inhibited by diltiazem (20-mu-M). These findings showed that voltage-dependent calcium channels are not functional in DDT1 MF-2 smooth muscle cells in contrast to freshly obtained Syrian hamster vas deferens smooth muscle. It is concluded that at least two populations of voltage-dependent channels are present in DDT1 MF-2 smooth muscle cells, conducting a slow outward rectifying current carried by potassium, and a fast, inactivating sodium current.