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.
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