Exposure of detergent-solubilized MthK channels at pH 9.0 in the nominal absence of Ca²⁺ results in gradual depletion of docked RCK domains from the channel complex. (A) Elution profile of purified detergent-solubilized MthK at pH 6.8 from a Superdex 200 size-exclusion column. Solutions contained 200 mM KCl, 5 mM DM, 10 mM HEPES, 2 mM nitrilotriacetic acid, and either 0 or 20 mM of added Ca²⁺. Under these conditions, MthK eluted primarily as a single peak centered at ∼10.5 ml either with or without Ca²⁺ (blue and red traces, respectively), with a relatively small peak at ∼15 ml (indicated by arrow) corresponding to monomeric RCK domain. (B) Elution profile of purified detergent-solubilized MthK at pH 9.0. Solutions were identical to those in A, except that 10 mM CHES was used instead of HEPES. With 20 mM Ca²⁺ (blue trace), MthK eluted primarily as a single peak centered at ∼11 ml. With nominally 0 Ca²⁺ (red trace), MthK eluted with a major peak centered at ∼10.5 ml and a peak at ∼15 ml that was larger than that observed at pH 6.8. (C) A series of size-exclusion chromatography runs at pH 9.0 with nominally 0 Ca²⁺, in which fractions eluting between 9 and 14 ml were pooled, concentrated, and reloaded. With each successive run, the peak at ∼15 ml is diminished, consistent with depletion of dissociated RCK domains. In addition, two peaks are resolved from the original single peak at ∼10.5 ml (a) and 12 ml (b). (D) Analysis of pooled fractions eluting between 9 and 14 ml from the first and fourth successive chromatography runs by SDS-PAGE, stained with Coomassie blue. Fractions from the first run show bands at ∼200 and 26 kD, consistent with elution of a channel complex containing transmembrane and soluble RCK subunits, as shown previously (Jiang et al., 2002a; Parfenova et al., 2006). Fractions from peaks (a) and (b) in the fourth run contain the ∼200-kD band but are depleted of the 26-kD band. This is consistent with peaks (a) and (b) each being comprised primarily of transmembrane subunits of the MthK channel that are depleted of “soluble” RCK subunits.