Strategy to determine the PUFA action site. (A) Sequence of segment S3–S6 for the Shaker K channel. //, the extracellular linkers are omitted; *, tested residues. Underlined residues mark helical transmembrane segments. (B) Structures of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). View from the extracellular side (left). Only one VSD and part of the pore domain are shown. View from the membrane side (right) as indicated by the arrow in the left panel. Only one VSD and the closest pore domain from another subunit are shown. Selectivity filter regions from all four subunits are displayed in cyan. The blue residues are the four most extracellular gating charges in S4 (R362, R365, R368, and R371). Red residues are explored in the present investigation. (C) Only the negatively charged form of the carboxyl group affects the voltage sensor. The effect is pH dependent. The introduction of a fixed positive charge close to the PUFA changes the local pH, deprotonates the carboxyl group, and potentiates the PUFA effect on the voltage sensor. The closer the charge is to the PUFA, the larger the potentiation is.