Structural and Energetic Basis of Ca²⁺-Selective Permeation in the TRPV5 Channel

TRPV5 is a calcium-selective epithelial ion channel that is essential for renal Ca²⁺ reabsorption and systemic calcium homeostasis. Despite recent structural advances, the energetic basis and coordinated contributions of the three selectivity-filter residues (D542, T539, and N572/I575) to Ca²⁺ selectivity remain unresolved. Here, we combine absolute and relative binding free-energy calculations (ABFE/RBFE), pore radius, and electrostatic analyses, and adaptive steered molecular dynamics (ASMD) to quantify how Ca²⁺ and Na⁺ interact with and traverse these key sites in both wild-type and mutant channels. Free-energy analyses show that all three sites favor Ca²⁺ over Na⁺, with the outer D542 site making the dominant contribution to Ca²⁺ selectivity, whereas T539 and N572/I575 provide weaker secondary contributions. ASMD-derived free-energy profiles further reveal a pronounced energetic preference for the Ca²⁺-driven displacement of Na⁺. Mutation of any one of the three selectivity-filter residues induced perturbations of pore radius and electrostatic landscapes attenuated the energetic preference, revealing the cooperative contribution of these three sites to Ca²⁺ selectivity in wild-type TRPV5. Based on the above results, we provide a hierarchical mechanism involving high-affinity Ca²⁺ capture at D542 and subsequent downstream modulation of the free-energy landscape by T539 and N572, forming an integrated structure-energy framework for selective Ca²⁺ permeation, offering testable hypotheses for disease variants and targeted modulation of TRPV5 in calcium-handling disorders.