BK α and β1 subunits. (A) N-terminal 350 residues of mouse BK α (full-length 1,169 residues) with mutations and insertions above the original sequence. pWTa has an N-terminal insertion (at slash mark) of the HA epitope (underlined), followed by a linker with extra Lys as targets for surface biotinylation. Cys14 and Cys141 were mutated to Ala, and the intracellular Cys residues in the S0–S1 loop, Cys53, Cys54, and Cys56, were mutated to Ser. pWTb also has an insertion of an HRV-3C protease consensus site (underlined) with linkers after S6 (at the slash mark). pWTc has a second HRV-3C site (underlined) in the S0–S1 loop, starting at residue A89L. The extracellular residues flanking the TM helices that we previously mutated to Cys (Liu et al., 2008a) are in white letters on black; the predicted intracellular flanking residues are highlighted in red; and residues, the mutation of which to Cys yielded functional channels, are in white, and those that yielded nonfunctional channels are in black. (B) Sequence of mouse β1 with insertions and mutations in the background construct. pWT β1, in which Cys18 and Cys26 are mutated to Ala. FLAG-HIS-pWT β1 has FLAG and HIS epitopes and linker at the N terminus of pWTβ1, and the mutation E13Q. TM1 and TM2 are highlighted in cyan. The extracellular flanking residues mutated to Cys are in white letters on a black background (Liu et al., 2008b); the intracellular flanking residues mutated to Cys are in white on a red background. (C) Membrane topology of BK α showing the residues in the predicted intracellular flanks of the TM helices. The HRV-3C protease cleavage sites are shown. (D) Membrane topology of BK β1 showing residues mutated to Cys in the intracellular flanks of the TM helices. Cys18 and Cys26 within TM1 were mutated to Ala. (E) Table of α background constructs.