A co-operative knock-on mechanism underpins Ca ²⁺ -selective cation permeation in TRPV channels

The selective exchange of ions across cellular membranes is a vital biological process. Ca ²⁺ -mediated signalling is implicated in a broad array of physiological processes in cells, whilst elevated intracellular concentrations of Ca ²⁺ are cytotoxic. Due to the significance of this cation, strict Ca ²⁺ concentration gradients are maintained across the plasma and organelle membranes. Therefore, Ca ²⁺ signalling relies on permeation through selective ion channels that control the flux of Ca ²⁺ ions. A key family of Ca ²⁺ -permeable membrane channels are the polymodal signal-detecting Transient Receptor Potential (TRP) ion channels. TRP channels are activated by a wide variety of cues including temperature, small molecules, transmembrane voltage and mechanical stimuli. Whilst most members of this family permeate a broad range of cations non-selectively, TRPV5 and TRPV6 are unique due to their strong Ca ²⁺ -selectivity. Here, we address the question of how some members of the TRPV subfamily show a high degree of Ca ²⁺ -selectivity whilst others conduct a wider spectrum of cations. We present results from all-atom molecular dynamics simulations of ion permeation through two Ca ²⁺ -selective and two non-selective TRPV channels. Using a new method to quantify permeation co-operativity based on mutual information, we show that Ca ²⁺ -selective TRPV channel permeation occurs by a three binding site knock-on mechanism, whereas a two binding site knock-on mechanism is observed in non-selective TRPV channels. Each of the ion binding sites involved displayed greater affinity for Ca ²⁺ over Na ⁺ . As such, our results suggest that coupling to an extra binding site in the Ca ²⁺ -selective TRPV channels underpins their increased selectivity for Ca ²⁺ over Na ⁺ ions. Furthermore, analysis of all available TRPV channel structures shows that the selectivity filter entrance region is wider for the non-selective TRPV channels, slightly destabilising ion binding at this site, which is likely to underlie mechanistic decoupling.