External ATP triggers a biphasic activation process of a calcium-dependent K+ channel in cultured bovine aortic endothelial cells

We have used the patch-clamp method in order to investigate the single-channel events underlying the effect of external ATP on the potassium permeability of bovine aortic endothelial cells (BAE). The results obtained from cell-attached and inside-out experiments led first to conclude that BAE cells possess an inward rectifying potassium channel activated by internal calcium at micromolar concentrations. The channel conductance for inward currents was estimated at 40 pS in symmetrical 200 mM KCl and the open-channel probability was found to be voltage insensitive within the membrane voltage range –50 to –100 mV. Based on results obtained in the cell-attached configuration, it could next be established that external ATP and ADP at micromolar concentrations could trigger, via the stimulation of P₂ purinergic receptors, a time variable activation process of the observed calcium-dependent potassium channel. This activation process was found to occur in a biphasic manner with an initial phase independent of the presence of calcium in the cell bathing medium. The second phase which could be blocked by calcium channel blockers such as Co²⁺ or La³⁺ required, however, the presence of external calcium and could be abolished by depolarizing the cells using high K⁺ external solutions. Another important aspect related to this phenomenon was the observation that removing ATP from the external medium during the second phase led to a complete abolition of the associated calcium-dependent potassium channel activation process. It is suggested from these results that the action of ATP on the potassium permeability of BAE cells is related to a second messenger mediated release of calcium from internal calcium stores coupled to an ATP-dependent calcium influx abolished at depolarizing voltages.