Concentration‑dependent switching between ligand‑like and solvent‑like lipid regulation in a model ion channel

Abstract Cell membranes are not only protective barriers but also active chemical environments that regulate protein structure and function. However, how individual lipid species influence membrane proteins across physiological concentration ranges, either as specific ligands or through bilayer-mediated solvent effects, remains poorly defined. Here, using the cyclic nucleotide-gated ion channel SthK reconstituted into defined membranes, we show that phosphatidic acid enhances channel activity across all tested concentrations but, at low mole fractions ( ~ 0.7-1 mol%), it selectively and cooperatively increases agonist potency, meeting criteria for a site‑specific allosteric ligand. In contrast, the zwitterionic lipid phosphatidylethanolamine modulates SthK activity exclusively through linear, solvent‑like effects across the same concentration range. Structural analysis reveals that low anionic lipid content expands the closed‑state conformational ensemble without promoting pore opening, providing a structural basis for lipid-dependent latent allostery. Together, these results establish lipid concentration as a governing regulatory variable that quantitatively distinguishes ligand‑like from solvent‑like mechanisms of membrane protein regulation.