Asymmetry of Rb⁺ conduction emerged under bi-ionic conditions in epithelial maxi-K⁺ channels

K⁺ channels permit more than one ion within their conducting pathway at any given moment and show a saturating single-file behavior. The conduction of Rb⁺ shows an unusual behavior, a so-called "Rb⁺ anomaly," and it has been used to probe the mechanism of the ion conduction through K⁺-selective channels. Under the bi-ionic condition of K⁺ and Rb⁺, we carried out patch-clamp single-channel current measurements in MaxiK⁺ channels from mouse submandibular acinar cells. Keeping only K⁺ on one side of the membrane while varying fractional Rb⁺ concentration on the opposite, we had a series of current-voltage relationships. It showed a characteristic inflection at which the ion conductance was divided into two components, one ascribed to pure K⁺ conduction and the other to K⁺ and Rb⁺ bi-ionic conduction. By analyzing the latter, we depicted that (1) the bi-ionic conductance showed a characteristic reduction curve as the Rb⁺ fractional concentration increased; (2) Rb ⁺ can bind the channel more tightly when it accesses from the outside than from the inside. Thus we conclude that such asymmetry of the Rb ⁺ binding determines the pattern of bi-ionic conductance reduction in K-selective channels.