Patch-Clamp Study of the Ionic Currents Underlying Action Potentials in Cultured Frog Pituitary Melanotrophs

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Abstract
The ionic conductance mechanisms underlying the action potential behaviour of frog melanotrophs in primary culture were studied by using the patch-clamp technique in whole-cell configuration. The action potentials spontaneously generated by these cells were predominantly sodium spikes with a calcium component. Voltage-dependent sodium, calcium, potassium and calcium-activated potassium currents were identified and analysed separately. The voltage-dependent sodium current was characterized by its fast kinetic, its low-threshold activation, its voltage-dependent inactivation and a tetrodotoxin sensitivity. Calcium currents were identified on the basis of their ionic selectivity to divalent cations (Ba2+, Ca2+, Co2+) and their time course. Only two of the three well-documented calcium currents could be detected in frog melanotrophs. A sustained calcium current (IcaS) and an inactivating calcium current (IcaN) were elicited by step depolarizations up to -20 mV. IcaN inactivated for membrane potentials more positive than -50 mV; its inactivation appeared to be both voltage- and calcium-dependent. Transient calcium current (IcaT) has never been observed. Two types of potassium currents were identified: voltage-dependent potassium (IĸV) and calcium-activated potassium currents, (Iĸ[Ca]). They were both suppressed by tetraethylammonium chloride, whereas only Iĸ(Ca) was blocked by cobalt. These major ionic currents underlying spontaneous electrical activity are assumed to be involved in the process of α-melanocyte-stimulating hormone release. The present study provides the ground for future investigations regarding the relationships between the electrical and secretory activities in amphibian pars intermedia cells.

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