Abstract
Calcium (Ca2+) is an essential second messenger required for diverse signaling processes in immune cells. Ca2+ release-activated Ca2+ (CRAC) channels represent one main Ca2+ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the endoplasmic reticulum, and the Ca2+ ion channel Orai in the plasma membrane. After Ca2+ store depletion, STIM1 and Orai couple to each other, allowing Ca2+ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca2+ selectivity, an increase in current density when switching from a Ca2+-containing solution to a divalent-free Na+ one, and fast Ca2+-dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly, these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct from those that controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N terminus is essential for STIM1, as it fine-tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.
Highlights
Calcium (Ca2؉) is an essential second messenger required for diverse signaling processes in immune cells
In our study, which focused on these three Ca2؉ release-activated Ca2؉ (CRAC) channel hallmarks, we discovered that several constitutively active Orai1 and Orai3 mutants displayed authentic CRAC channel activity, but only in the presence of stromal interaction molecule 1 (STIM1) and the conserved portion of the Orai N terminus
One of the major hallmarks of CRAC channel permeation is the increase in current density when switching from a Ca2ϩcontaining to a DVF Naϩ-containing solution, as exemplified for wildtype Orai1 as well as wildtype Orai3, each co-expressed with STIM1 (Fig. S1)
Summary
One of the major hallmarks of CRAC channel permeation is the increase in current density when switching from a Ca2ϩcontaining to a DVF Naϩ-containing solution, as exemplified for wildtype Orai as well as wildtype Orai, each co-expressed with STIM1 (Fig. S1). The permeation characteristics of Orai F160A when switching from an extracellular to a DVF solution exhibited a dependence on STIM1, with a current decrease and increase in the absence and presence of STIM1, respectively. This indicated that the persistence of this CRAC channel hallmark was not dependent on the duration of the applied voltage ramp (Fig. S5). The further deletion of two additional N-terminal residues (i.e. Orai1 ⌬N1–74 L185A/F250A) led to a loss of these CRAC channel hallmarks in the presence of STIM1, resulting in substantially decreased current responses to DVF solution concomitant to a strong current potentiation (Fig. 5, a– e). Maintenance of the typical CRAC channel hallmarks required the first half of the conserved N-terminal region, whereas the second half was imperative for store-operated CRAC channel activation
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