Protein–lipid interplay governs ion channel gating and bioenergetics in human mitochondrial <scp>VDAC3</scp>

The interaction between transmembrane proteins and their lipid environment is central to protein stability and function. Yet, the molecular factors guiding specificity of protein-lipid interactions remain poorly defined. Here, using the human voltage-dependent anion channel 3 (hVDAC3), a member of the essential outer mitochondrial membrane β-barrel VDAC family, as a model, we show that lipid headgroups, and hydrocarbon chain length and saturation, critically shape ion channel behavior. Physiological lipids effectively mitigate the functional deficiency of hVDAC3. Surprisingly, cardiolipin uniquely disrupts hVDAC3 gating by preferentially retaining the channel in an "open-like" conductive state. Single-channel electrophysiology and all-atom molecular dynamics simulations together reveal that lipid composition selectively modulates hVDAC3 structure and its N-terminal helix dynamics, without altering the global β-barrel fold and stability. We conclude that anionic headgroups, negative protein-bilayer mismatch, and increased membrane viscosity favor optimal channel stability and function. We find the specific functional outcome of cardiolipin-hVDAC3 cross-talk a potential regulator of the onset of mitochondria-mediated apoptosis. These findings offer fundamental insights into the unexpected sensitivity of mitochondrial channels to the physicochemical diversity of their lipid environment.