Two barium binding sites on a maxi K⁺ channel from human vas deferens epithelial cells

Using the patch clamp technique, we have investigated the blockade of maxi-K⁺ channels present on vas deferens epithelial cells by extracellular Ba²⁺. With symmetrical 140 mM K⁺ solutions, Ba²⁺ produced discrete blocking events consisting of both long closings of seconds duration (slow block) and fast closings of milliseconds duration (flickering block). Kinetic analysis showed that flickering block occurred according to an 'open channel blocking' schema and was eliminated by reducing external K⁺ to 4.5 mM. Slow block showed a complex voltage-dependence. At potentials between -20 mV and 20 mV, blockade was voltage-dependent; at potentials greater than 20 mV, blockade was voltage-independent, but markedly sensitive to the extracellular K⁺ concentration. These data reveal that the vas deferens maxi-K⁺ channel has two Ba²⁺ binding sites accessible from the extracellular side. Site one is located at the cytoplasmic side of the gating region and binding to this site causes flickering block. Site two is located close to the extracellular mouth of the channel and binding to this site causes slow block.