Molecular determinants within N terminus of Orai3 protein that control channel activation and gating.

STIM1 and Orai represent the key components of Ca(2+) release-activated Ca(2+) 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.

Cooperativeness of Orai cytosolic domains tunes subtype-specific gating.

Activation of immune cells is triggered by the Ca(2+) release-activated Ca(2+) current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca(2+) overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, whereas Orai2 and Orai3 currents display a second, slow inactivation phase.

Plasticity in Ca2+ selectivity of Orai1/Orai3 heteromeric channel.

A general cellular response following depletion of intracellular calcium stores involves activation of store-operated channels (SOCs). While Orai1 forms the native Ca(2+) release-activated Ca(2+) (CRAC) channel in mast and T cells, the molecular architecture of less Ca(2+) selective SOCs is insufficiently defined. Here we present evidence that diminished Ca(2+) selectivity and robust Cs(+) permeation together with a reduced fast inactivation are characteristics of heteromeric Orai1 and Orai3 channels in contrast to their homomeric forms.

Molecular determinants of the coupling between STIM1 and Orai channels: differential activation of Orai1-3 channels by a STIM1 coiled-coil mutant.

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca(2+) release-activated Ca(2+) current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by approximately 50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner.

Increased hydrophobicity at the N terminus/membrane interface impairs gating of the severe combined immunodeficiency-related ORAI1 mutant.

Patients with severe combined immune deficiency (SCID) suffer from defective T-cell Ca2+ signaling. A loss of Ca2+ entry has been linked at the molecular level to single missense mutation R91W in the store-operated Ca2+ channel ORAI1. However, the mechanistic impact of this mutation on ORAI1 function remains unclear.

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels.

In immune cells, generation of sustained Ca(2+) levels is mediated by the Ca(2+) release-activated Ca(2+) (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca(2+) sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca(2+) stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels.

The STIM/Orai coupling machinery.

Calcium (Ca(2+)) entry into non-excitable cells is mainly carried by store-operated channels (SOCs), which serve essential functions ranging from regulation of transcription to cell growth. The best-characterized store-operated current, I(CRAC), is the calcium release-activated calcium (CRAC) current initially discovered in T-lymphocytes and mast cells. The search for the molecular components of the CRAC channel lasted over 20 years.

2-aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size.

Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca(2+) release-activated Ca(2+) channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 microm 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents.

Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation.

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction.