Orai1 Mutations Alter Ion Permeation and Ca²⁺-dependent Fast Inactivation of CRAC Channels: Evidence for Coupling of Permeation and Gating

Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca²⁺ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca²⁺ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca²⁺ block, and channel regulation by Ca²⁺. We find that alteration of the acidic residues lowers Ca²⁺ selectivity and results in striking increases in Cs⁺ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs⁺ permeation. Ca²⁺ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca²⁺ binding. Unexpectedly, the mutations diminish Ca²⁺-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca²⁺ selectivity, increase in Cs⁺ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.