Ion permeation mechanism in epithelial calcium channel TRVP6

Based on TRPV6 crystal structures, molecular dynamics simulations predicted binding sites for calcium, barium and gadolinium inside the channel pore. The first TRPV6 crystal structure was solved in our lab in 2015 and published in 2016 in Nature. We succeeded to determine several crystal structures of the channel bound to Ca²⁺, Ba²⁺ and Gd³⁺ and identified ion binding sites in the channel pore as well as in the extracellular vestibule (recruitment sites). Site locations and ion coordination geometry allowed us to make initial assumptions about ion permeation mechanism in TRPV6. It is well known that at low Ca²⁺ concentrations, the TRPV6-mediated inward current is mainly carried by Na⁺ ions. An increase in Ca²⁺ concentration leads to an increase in Ca²⁺ current but a reduction of Na⁺ current. We used MD simulations with varying numbers of Ca²⁺ and Na⁺ ions to verify experimental observations and to test our structural models.

Briefly, at low Ca²⁺ concentrations, only a single calcium ion binds at the narrow constriction of the TRPV6 selectivity filter formed by aspartates D541 and allows Na⁺ permeation. During ion permeation, no water crosses the channel pore narrow constriction. At high Ca²⁺ concentrations, calcium permeates the pore according to the knock-off mechanism that includes formation of a short-lived transition state with three calcium ions bound near D541. For Ba²⁺, the transition state lives longer and the knock-off permeation occurs slower. Gd³⁺ binds at D541 more tightly, blocks the channel and prevents Na⁺ from permeating the pore.

Movie1. Knock-off mechanism of Ca²⁺ permeation. Protein is shown in cyan cartoon representation. The incoming, intermediate, and leaving Ca²⁺ ions are shown as blue, magenta, and brown spheres respectively, other Ca²⁺ ions are show as green spheres. D541 and T538 are shown as yellow sticks with oxygen and nitrogen in red and blue respectively. Ca²⁺ permeates via knock-off mechanism. The total length of the simulation shown is ca. 30 ns.

Movie 2. Na⁺ permeation through TRPV6 channel. Protein is shown in cyan cartoon representation, Na⁺ ions are shown as white spheres, and D541, T538, M569 are shown as yellow sticks with oxygen and nitrogen in red and blue respectively. Multiple Na⁺ ions occupied the selectivity filter. The total length of the simulation shown is ca. 30 ns.

This work was done in collaboration with Dr. Kurnikova’s lab from Carnegie Melon University.