Cu²⁺ triggers lipid phase separation in anionic membranes via a bimodal interfacial mechanism

The cellular membrane serves as the primary interface for sensing environmental cues, yet how it decodes the concentration-dependent toxicity of essential metal ions like copper remains a fundamental question. Here, we unveil a bimodal molecular switching mechanism by which Cu²⁺ reorganizes anionic lipid membranes, suggesting that the cell membrane could act as a sensor for copper concentration. Using an integrative biophysical approach on phosphatidylcholine/phosphatidylglycerol membranes, we demonstrate that at low concentrations, Cu²⁺ binds individually to anionic lipids, reorienting headgroups and priming the membrane for separation (Stage I). Crucially, beyond a critical threshold, adjacent membrane-bound Cu²⁺ ions form metal-metal bonds, creating rigid [PG-Cu-Cu-PG] bridges that act as molecular clamps (Stage II). This cooperative bridging event forcefully squeezes lipids together, driving extensive phase separation and the formation of Cu-rich domains with markedly enhanced thermodynamic stability. We provide evidence through atomic force microscopy, X-ray scattering, and calorimetry, complemented by the spectroscopic signature of ESR-silent Cu-Cu pairs. This bimodal switch model provides a physicochemical basis for copper's dual biological identity, with direct implications for understanding its antimicrobial mechanism and its potential role in neurotoxicity. Our findings establish a new paradigm for how transition metal coordination chemistry can programmatically control membrane architecture.