Existence of calcium channels and intercellular couplings in the testosterone-secreting cells of the mouse.

1. The electrophysiological properties of testosterone-secreting cells (i.e. Leydig cells) in the mouse were studied using patch electrodes. The cells appeared solitarily or in clusters after mechanical dissociation from testes. They were confirmed to be Leydig cells on the basis of 3 beta-hydroxysteroid dehydrogenase staining. 2. Under current-clamp conditions in the whole-cell configuration, Leydig cells immersed in standard saline were able to generate action potential-like responses. The active responses occurred after cessation of membrane hyperpolarization or when cells were held in a hyperpolarized condition and stimulated with depolarizing current pulses. 3. In Leydig cells under voltage clamp, depolarizations more positive than -50 mV evoked transient inward currents which decayed completely during the duration of depolarization (130 ms). No obvious outward currents were evoked by pulses less positive than 30 mV. 4. The inward currents were identified as Ca2+ current, since replacement of external Ca2+ with Mn2+ reversibly diminished the current whereas Ba2+ or Sr2+ substituted for Ca2+. 5. With voltage pulses more positive than 40 mV, outward currents were evoked. The currents were dependent on K+ concentration and were blocked by quinine or tetraethylammonium. The amplitudes of outward currents were increased with raised internal Ca2+ concentration. 6. Single-channel recordings of the outward currents revealed that the unitary conductance was 130 pS when internal K+ was 131-143 mM and external K+ was 5 mM. The open probability of the channel showed marked dependence on the membrane potential and the internal Ca2+ concentration. Thus, the current was identified as being Ca2+- and membrane potential-dependent K+ current. 7. Leydig cells within a cluster possessed distinct intercellular couplings. The mean coupling ratio obtained by applying two patch electrodes to a pair of cells was 0.84. Transfer of injected dye (Lucifer Yellow) to adjacent cells was also confirmed. 8. It was concluded that Leydig cells have at least two kinds of voltage-dependent channels in the membrane. The Ca2+ channel may be activated by physiological changes in membrane potential, leading to an influx of Ca2+. The Ca2+-dependent K+ channel hardly seems to be activated unless the internal Ca2+ concentration increases remarkably. It is presumed that intercellular coupling may play a role in synchronizing or intensifying the endocrine activities of Leydig cells located within a cluster.