Changes in VSD and VSD-RCK1 interactions with ligation. (A) Comparison between liganded (cyan) and metal-free (gray) aSlo1 structures in the membrane-spanning domain for a single subunit. Structures were aligned such that selectivity filter and the pore helix (left of the structure) are superimposed. Side chains that contribute to gating charges (labeled with mSlo1 numbering) are shown in red ball-and-chains, with dotted lines helping identify some residues. S3 is hidden under S2. (B) Portion of A showing only S2, S3, and S4 for liganded (cyan) and metal-free (gray) structures, highlighting rather modest changes in VSD between structures. R213 corresponds to primary S4 gating charge residue. (C) Side chains of various mSlo1 (D153, D186, R167, R213, R219) and Kv1.2 (E226, R303, R309) implicated in voltage-sensing are shown for metal-free and liganded structures. Distance in angstroms is listed for net movement of charge center of given residue; dotted lines show vector of displacement for specific residues. Kv1.2 R309, although not a voltage-sensing residue, is shown to emphasize large extent of motion in S4 of Kv1.2. R213 in Slo1 and R303 of Kv1.2 correspond to the identical R4 position. The aSlo1 and Kv1.2 structures were aligned based on the selectivity filters, but the corresponding K⁺ ion positions have been displaced closer to the S4 positions to minimize figure size. (D) Part of two adjacent subunits of the liganded aSlo1 structure are displayed, one (gray) showing S1–S6 and the other (green) showing part of S6 and N-lobe of RCK1. Residue labeling corresponds to mammalian sequence; mE219/aD208 and mR213/aR202 in S4, mK234/aR223 in S5, and mE321/mE324 (aE310/aE313) in the C-linker are shown in red in one subunit; mE321 and mE324 in the other subunit are shown in blue. Residues involved in Mg²⁺ coordination are also shown, along with two residues (mD99/aD88 and mK342/aK331) that may stabilize an interaction between the TM domain and the N-lobe. Dotted vertical line highlights the pore axis along with K⁺ ions (not part of structure). The αB helix of the N-lobe suggested to be involved in interactions with TM elements is also highlighted in red. (E) Liganded structures for the same elements shown in C are shown, showing the pronounced repositioning of the N-lobe of one subunit to allow interaction with the elements of the transmembrane domain. The S6 helix associated with the subunit containing the N-lobe appears to shift approximately one helical turn once the N-lobe interaction is stabilized. The panel highlights several potential domain interactions that may influence gating, including (1) interactions among S4–S5 with S6, (2) interactions among VSD and the S4–S5 linker with the N-lobe of RCK1, (3) interactions between mD99 and mK342, and (4) direct connection via the C-linker between the N-lobe of RCK1 and S6.