A novel type of internal barium block of a maxi-K⁺ channel from human vas deferens epithelial cells

We have recently shown that a maxi-K⁺ channel from vas deferens epithelial cells contains two Ba²⁺-binding sites accessible from the external side: a 'flickering' site located deep in the channel pore and a 'slow' site located close to the extracellular mouth of the channel. Using the patch-clamp technique, we have now studied the effect of internal Ba²⁺ on this channel. Cytoplasmic Ba²⁺ produced a voltage- and concentration- dependent 'slow' type of block with a dissociation constant of ~100 μM. However, based on its voltage dependence and sensitivity to K⁺ concentration, this block was clearly different from the external 'slow' Ba²⁺ block previously described. Kinetic analysis also revealed a novel 'fast flickering' block restricted to channel bursts, with an unblocking rate of ~310 s^-1, some 10-fold faster than the external 'flickering' block. Taken together, these results show that this channel contains multiple Ba²⁺-binding sites within the conduction pore. We have incorporated this information into a new model of Ba²⁺ block, a novel feature of which is that internal 'slow' block results from the binding of at least two Ba²⁺ ions. Our results suggest that current models for Ba²⁺ block of maxi-K⁺ channels need to be revised.