Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

Abstract

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of −62±3 mV (n=8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na⁺-free bath and were reversibly blocked by 4 mM Co²⁺. They lasted 0.9–3.0 s with 64 mM Cl⁻ in the pipette, were shortened 67±8 % by the Cl⁻ channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 20 μM), and lengthened to 8.7±2.2 when the Cl⁻ equilibrium potential (V _Cl) was changed from −20 mV to −2 mV by using 134 mM Cl⁻ⁱⁿ the pipette. With conventional whole-cell voltageclamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35–1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of −60 mV and −50 mV and reached a maximum inward amplitude at about −40 mV. Changing the Cl⁻ concentration in the pipette (V _Cl of −2 mV or −20 mV) or bath (V _Cl of −2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl⁻ electrode. They were inhibited by the Cl⁻ channel antagonists 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 0.5 mM), NPPB (20 μM), and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca²⁺ deprivation, or by Co²⁺ (4 mM), or by replacing external Ca²⁺ with Ba²⁺. They showed pronounced inward rectification when a weakly buffered 0.2 μM Ca²⁺ pipette solution was used, but this rectification was much reduced when pipette solutions contained 5 μM Ca²⁺. The function of this Ca²⁺-dependent Cl⁻ current and the physiological trigger(s) of the action potentials and their role(s) in granulosa cell function remain to be determined.