Removing the ring four negative charges (Glu322) in the external mouth of the BK channel induces outward rectification. (A) Single channel current measured as a function of membrane potential for mutant E322I (black dots and solid line) compared with the WT channels (red line) measured in symmetrical 110 mM K-MES and 1 M K-MES. (B) Column plot of the average single channel conductance measured at −100 mV for the control and double mutant channels at 110 mM K-MES and 1 M K-MES. (C) Electrostatic potential calculated associated with turning off the charges of residue Glu322.