Negatively charged amino acids located in the external loop have a marginal effect on single channel conductance. (A) Samples of single channel current fluctuations induced by the WT BK channel and the neutralization mutants and recorded at −100 mV in symmetric 110 mM K⁺. Current–voltage relationship for WT (B) and neutralization BK channel mutants (C–E). Single channel current was measured from the peaks of all-points histograms of the current intensity records of membranes containing 1, 2, or 3 channels. (C) Current–voltage relationship for the E329Q mutant. (D) Current–voltage relationship for the D326N mutant. (E) Current–voltage relationship for the D326N-E329Q double mutant. Experiments were performed at room temperature, 20–22°C.