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

Abstract

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.

Plasticity in Ca 2+ selectivity of Orai1/Orai3 heteromeric channel

“…2 E and F; n ϭ 10 cells). With an estimated permeability ratio P Cs /P Na ϭ 0.73 Ϯ 0.07 calculated by the Goldman-Hodgkin-Katz equation (29), the Orai3-Orai1 tandem is dramatically less selective for Na ϩ over Cs ϩ under divalent-free conditions in comparison to 0.18 Ϯ 0.02 for Orai1 and 0.11 Ϯ 0.02 for Orai3 in line with a previous calculation of 0.15 for Orai1 currents (24). Native CRAC channels (30) in Jurkat T-cells yielded P Cs /P Na ϭ 0.13.…”

“…The narrowest part of the Orai1 and Orai3 pore is approximately 3.8 Å (23,24), suggesting that steric hindrance most likely accounts for low Cs ϩ permeability in homomeric Orai channels. Therefore, increased Cs ϩ permeability and consequently reduced Ca 2ϩ selectivity is a fingerprint of the heteromeric assembly of Orai1 and Orai3 proteins.…”

“…1H), as well as Orai3 channels (24) are permeable to sodium in a DVF solution (Na ϩ -DVF), yielding an inward rectifying current-voltage relationship that reversed at ϩ43 Ϯ 3 mV (n ϭ 6 cells) and ϩ46 Ϯ 5 mV (n ϭ 4 cells), respectively. Co-expression of Orai1 and Orai3 together with Stim1 led to an inward rectifying current-voltage relationship in Na ϩ -DVF solution with a remaining outward component that reversed at ϩ32 Ϯ 7 mV ( Fig.…”

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…Srikanth et al suggest that the II-III loop of Orai1 may function as a blocking particle in CDI to inhibit conduction. Another study has shown that mutations that increase Orai1 pore size (E106D, E190Q) greatly reduce or eliminate CDI (Yamashita et al 2007). These findings raise the possibility that the inactivation gate may involve elements of the selectivity filter, as has been proposed for Ca V and K V channels.…”

Orai1 pore residues control CRAC channel inactivation independently of calmodulin

“…4 E and Table 1) of Orai1 Y80E suggest that in addition to abrogating CDI, this mutation also significantly alters the pore structure. The interaction of permeation and inactivation gating has been described before for the Orai1 E106D mutant channel, an outer pore mutant with markedly decreased Ca 2+ affinity together with decreased CDI (Yamashita et al, 2007). Thus, an altered structure of the outer pore near the selectivity filter in Y80E may permit greater Ca 2+ flux while simultaneously preventing a conformational change that underlies CDI.…”