Purinergic activation of BK channels in clonal kidney cells (Vero cells)

We have studied the activation of a high-conductance channel in clonal kidn ey cells from African green monkey (Vero cells) using patch-clamp recording s and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single -channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl . The channel is highly selective for K+ and activated by membrane depolari zation and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel ). Cell-attached recordings revealed that the channel is inactive in unstim ulated cells. Extracellular application of less than 0.1 muM ATP transientl y increased the cytosolic Ca2+ concentration ([Ca2+](i)) to about 550 nM, a nd induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at bot h the normal-resting potential and during membrane hyperpolarization. The i ncrease in [Ca2+](i) was owing to Ca2+ release from internal stores, sugges ting that Vero cells express G-protein-coupled purinergic receptors (P-2Y) mediating IP3-induced release of Ca2+. The P-2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the ra nk of agonist potency was ATP > > UTP greater than or equal to ADP. This re sult indicates the presence of both P-2Y1 and P-2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown t hat hypotonic challenge activates BK channels in both normal and clonal kid ney cells. The subsequent loss of KCl may be an important factor in cellula r volume regulation. Our results support the idea of an autocrine role of A TP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P-2Y receptors, subsequently increasing [Ca2+](i) and thus causing K+ efflux through BK channels.