Glu106 in the Orai1 pore contributes to fast Ca2+-dependent inactivation and pH dependence of Ca2+ release-activated Ca2+ (CRAC) current

Abstract

FCDI (fast Ca²⁺-dependent inactivation) is a mechanism that limits Ca²⁺ entry through Ca²⁺ channels, including CRAC (Ca²⁺ release-activated Ca²⁺) channels. This phenomenon occurs when the Ca²⁺ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca²⁺ sensor that communicates the Ca²⁺ load of the intracellular stores to Orai1, have been shown to regulate fast Ca²⁺-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca²⁺-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca²⁺-binding site that regulates fast Ca²⁺-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca²⁺-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na⁺ permeation through the E106D Orai1 pore by Ca²⁺ is diminished. These results suggest that Glu¹⁰⁶ inside the CRAC channel pore is involved in co-ordinating the Ca²⁺-binding site that mediates fast Ca²⁺-dependent inactivation.

Regulation of endogenous and heterologous Ca2+ release-activated Ca2+ currents by pH

“…Recently, the glutamate residue E106 in Orai1 has been identified to contribute to the extracellular pH dependence of Ca 2+ release-activated Ca 2+ (CRAC) channels [48]. Here, we demonstrate that two aspartate residues in the first extracellular loop of Orai1, close to E106, also contribute to the pH-induced changes of I CRAC .…”

“…Close to the glutamate residue in position 106 (E106), which has already been shown to mediate some pH dependence [48], the first extracellular loop of Orai1 reveals two additional negatively charged residues: aspartate residues D110 and D112. An Orai1 construct in which these two aspartates were mutated to uncharged alanine residues (D110/112A) demonstrated that these residues contribute to the ion selectivity and to the anomalous mole-fraction behavior of Orai1 channels [46].…”

“…Others predict a similar mechanism as assumed for L-type Ca 2+ channels via the protonation of negatively charged glutamate residues close to the channel pore [43,44]. The glutamate residue E106, which is known to contribute to the selectivity filter of multimeric Orai1 channels [45-47], appears to be involved in the pH dependence of heterologously expressed STIM1/Orai1 CRAC channels [48]. Mutation of E106 to aspartic acid in Orai1 (E106D) resulted in a reverse pH dependence compared to Orai1 wild type: while Orai1 is normally blocked upon external acidification, Orai (E106D) exhibits an increase in inward current at pH 5.1.…”

Targeting calcium signaling in cancer therapy

“…In particular, E106 accounts for Ca 2+ selectivity in Orai1 and can be blocked by extracellular protons 202 . This class of inhibitor includes SB01990, SPB06836, KM06293 and RH01882, which all present the capability to alter the pore geometry of Orai1 and diminishes SOCE 164 .…”

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1

“…2D) were ϳ0.75 for STIM1 WT and ϳ0.78 for STIM1 DQ. To compare Ca 2ϩ -dependent inactivation and potentiation of I CRAC mediated by STIM1 WT/Orai1 and STIM1 DQ/Orai1 over the entire voltage range, we constructed apparent P o curves from normalized tail currents at Ϫ100 mV as previously described (31,32). Transfected with similar S1/O1 DNA ratios, STIM1 WT and STIM1 DQ currents showed both potentiation (points above dashed line corresponding to the apparent P o at 80 mV, set to unity) and inactivation (points below dashed line) in 10 mM [Ca 2ϩ ] o (Fig.…”