BK channel topology and assembly. (A) Membrane topology of a BK α subunit (Wallner et al., 1996; Meera et al., 1997). Voltage-sensing charged residues D153 and R167 (S2), D186 (S3), and R213 (S4) (Ma et al., 2006) are indicated by their charge polarity. (B) The sequences of the human BK extracellular S1–S2 (S134–K146) and S3–S4 (N200–S202) linkers, which include positions pertinent to this work: cysteine substitutions for fluorescent labeling (in red: S135C and Y145C) and Trp introductions for fluorophore quenching (in blue: I138W and C141W; note that C141 is normally substituted to serine to prevent fluorophore conjugation). The native Trp at the extracellular tip of S4 (W203) is also in blue. (C) Top view of a potassium channel transmembrane domain (K_V1.2–2.1 chimera; Protein Data Bank accession no. 2R9R; Long et al., 2007) with the addition of S0 ideal α helices to resemble BK channels. The pore domain (green) contains a K⁺ ion and is surrounded by four VSD helix bundles (orange). (D) Suggested packing arrangement of the BK VSD helix bundle (Liu et al., 2010).