Mouse and human BK α and β subunits. (A–C) Membrane topology of BK α, β2, and β3 showing the residues mutated to Cys in the extracellular flanks of the TM helices. The native extracellular Cys residues of BK α, Cys14, and Cys141 were mutated to Ala to create the pWT α background. Extracellular Cys residues in the extracellular loop of β2 and β3 are shown. The four Cys residues within the extracellular loop that are conserved in all four β-subunit genes are marked with an asterisk. (D) Sequences of the predicted extracellular loops of human and mouse BK β subunits aligned with ClustalW. The Cys in each of the β-subunit genes are highlighted in white text on a black background, and the four conserved Cys are marked with an asterisk. β4-subunit residues that have been implicated in mediating resistance to ChTX (Gan et al., 2008) are highlighted in green. β2-subunit residues that have been implicated in mediating resistance to ChTX and imparting rectification to α (Chen et al., 2008) are highlighted in red. Lys69 in the β1 subunit, which cross-links to ChTX (Knaus et al., 1994a; Munujos et al., 1995), is highlighted in gray. Gain-of-function and loss-of-function β1 polymorphisms, E65K (Fernández-Fernández et al., 2004) and R140W (Seibold et al., 2008), are highlighted in blue. Residues in β1 implicated in promoting voltage-sensor activation and reducing intrinsic gating (Gruslova et al., 2012) are highlighted in purple. The numbering of Cys from 1–8 is based upon the Cys in β2, hβ3, and β4.