Mutants only partially represent characteristics of calcium-release-activated calcium channel gating ^†

Calcium-release-activated calcium (CARC) channels are one of the major pathways of calcium entry in non-excitable cells. Despite a decade or two of research, its regulatory mechanism is not yet thoroughly understood. The slow progress is due to the complexity of its pores (i.e., Orai) on one hand and the difficulty in capturing its regulatory complex on the other hand. As a result, possible gating mechanisms have often been speculated by exploring the structure and properties of constitutive open mutants. However, there is much debate about how they can truly reflect the gating of CRAC channels under physiological conditions. In the present study, we combined molecular dynamics simulations with free energy calculations to study three dOrai mutants (G170P, H206A, and P288A), and further calculated their current-voltage curves. Results show that these constructs adopt different approaches to maintain their conductive state. Meanwhile they have unique pore structures and distinctive rectification properties and ion selectivity for cations compared to wild-type pores. We conclude that although the mutants may partially capture the gating motion characteristics of wild-type pores, the information obtained from these mutants is likely not a true reflection of CRAC channel gating under physiological conditions.