Abstract
STIM1 and Orai represent the key components of Ca2+ release-activated Ca2+ channels. Activation of Orai channels requires coupling of the C terminus of STIM1 to the N and C termini of Orai. Although the latter appears to be central in the interaction with STIM1, the role of the N terminus and particularly of the conserved region close to the first transmembrane sequence is less well understood. Here, we investigated in detail the functional role of this conserved region in Orai3 by stepwise deletions. Molecular determinants were mapped for the two modes of Orai3 activation via STIM1 or 2-aminoethoxydiphenyl borate (2-APB) and for current gating characteristics. Increasing N-terminal truncations revealed a progressive decrease of the specific fast inactivation of Orai3 concomitant with diminished binding to calmodulin. STIM1-dependent activation of Orai3 was maintained as long as the second half of this conserved N-terminal domain was present. Further truncations abolished it, whereas Orai3 stimulation via 2-APB was partially retained. In aggregate, the N-terminal conserved region plays a multifaceted role in Orai3 current gating with distinct structural requirements for STIM1- and 2-APB-stimulated activation.
Highlights
Ca2ϩ is a multifunctional messenger that regulates a variety of processes like muscle contraction, gene expression, proliferation, cell growth, and cell death
Deletion of Whole N Terminus of Orai3 Disrupts Its Activation—Because Orai3 channels can be stimulated by store depletion as well as 2-aminoethoxydiphenyl borate (2-APB), we utilized both modes of activation to investigate the role of the N terminus in the Orai3 gating machinery
Distinct molecular determinants were identified for Orai3 activation by STIM1 and 2-APB
Summary
Ca2ϩ is a multifunctional messenger that regulates a variety of processes like muscle contraction, gene expression, proliferation, cell growth, and cell death. Upon depletion of endoplasmic reticulum Ca2ϩ stores, STIM1 multimerizes and redistributes into punctae in close proximity to the plasma membrane [6, 8, 9] It co-localizes with the CRAC channel component Orai, culminating in channel activation followed by Ca2ϩ influx [7, 8, 10]. It has been reported recently that a single lysine within this conserved region (K85E in Orai and K60E in Orai3) represents a critipcal residue for store-operated activation [31]. This conserved region includes a binding site for CAD, a small Orai-activating STIM1 C-terminal fragment that interacts with higher affinity with the C terminus of Orai1 [30]. Gating Determinants within N Terminus of Orai residues in the N terminus of Orai are involved in the gating of the channel
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