Calcium-activated potassium channels in the luminal membrane of Amphiuma diluting segment: voltage-dependent block by intracellular Na+ upon depolarisation

Calcium-activated potassium channels in the luminal membrane of Amphiuma diluting segment were studied using the patch-clamp technique in both the cellattached and inside-out configurations. The open probability (P_o) of the channel is sensitive to both membrane potential and cytoplasmic calcium activity; depolarizing potenials and high calcium concentrations leading to an increased P_o. In the cell-attached condition, channel openings were observed between pipette potentials of –100 and –240 mV. As the driving force for potassium exit from the cell into the pipette is increased the single channel currents show a biphasic response. First, the currents increase as expected; however, the single channel currents diminish in magnitude at pipette potentials more negative than –120 mV. We propose that this reduction is due to rapid blockade of the potassium channel by intracellular sodium.This proposal is supported by two facts: (a) using inside-out patches it was possible to reduce the single channel currents in a concentration- and voltage-dependent manner, similar to that observed in the cell-attached condition, by raising the sodium concentration of the fluid bathing the cytoplasmic face of the patch; (b) pretreatment of tubules with the loop-acting diuretic furosemide (10^–5M), an agent known to decrease the intracellular sodium activity, caused an attenuation of the reduction in single channel current seen under control conditions. Given the very low P_o of the channels at the resting membrane potential and the sensitivity of the channels to intracellular sodium, it is unlikely that blockade of these channels by intracellular sodium would lead to a physiological regulation of the apical K conductance.