Extended Data Fig. 4: Functional assays using SthK.
From: Anionic lipids unlock the gates of select ion channels in the pacemaker family
(a) Quenching kinetics from a Tl+ flux assay reporting SthK channel activity in different lipid compositions (DOPC – black, 3:1 DOPC:POPG – orange, 3:1 DOPC:cardiolipin (CL) – blue) after 12 ms activation by 200 µM cAMP. Traces in the absence of cAMP are in shades of grey. (b). FSEC analysis (Superdex 200 5/150) of WT SthK extracted from liposomes as used for the stopped-flow assay (DOPC – black, 3:1 DOPC:X with X = POPE – dark blue, POPS – dark green, POPG – orange and POPA – red), excitation at 280 nm, emission at 330 nm. Signal intensity was normalized to SthK extracted from DOPC-only liposomes. (c) SDS-PAGE analysis of SthK reconstituted into different lipid compositions for the stopped-flow Tl+ flux assay is shown for WT SthK and SthK D226N. Detergent (DDM) or lipid environment for the reconstitution are indicated. DOPC and 3:1 DOPC:X were used for reconstitutions, with X = POPG (PG), POPE (PE), POPS (PS), POPA (PA), or cardiolipin (CL). Molecular weight standard used was Precision Plus Protein (Bio-Rad). (d) Comparison of the open probability (Po) values obtained from single-channel recordings (cf Fig. 2c, d). Averaged values ± s.d. (from Fig. 2d) are shown for WT SthK in the presence of POPE (dark blue), POPS (dark green), POPG (orange) and POPA (red). Mean Po values reach a stable non-zero level at negative voltages (inset). (e) Comparison of the single channel conductance values obtained from single channel recordings (cf Fig. 2c, d). Averaged values ± s.d. (from Fig. 2d) are shown for WT SthK in the presence of POPE (dark blue), POPS (dark green), POPG (orange) and POPA (red). (f) Fold-change in Po for SthK reconstituted into different lipid compositions at ±20 mV, shown relative to the value obtained in 3:1 DOPC:POPE. (g) Fold-change in single channel conductance I for SthK in different lipid compositions at ±20 mV relative to the value obtained in 3:1 DOPC:POPE. Lipid compositions for F and G are 3:1 DOPC:X, with X = POPE – dark blue, X = POPS – dark green, X = POPG – orange, X = POPA – red.
