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.
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