Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Abstract

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I CRAC ) [1][2][3][4] . However, individual overexpression of either protein fails to significantly amplify I CRAC . Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I CRAC , suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.Receptor-mediated release of Ca 2+ from intracellular stores induces Ca 2+ entry through calcium release-activated calcium (CRAC) channels [5][6][7] . Previous studies have identified STIM1 as the potential sensor for endoplasmic reticulum luminal Ca 2+ concentration 1,8,9 . When Ca 2+ is depleted from intracellular stores, STIM1 translocates to vesicular structures (punctae) underneath the plasma membrane, where it is hypothesized to activate CRAC channels residing in the plasma membrane. A second protein, CRACM1, has recently been identified as essential for activating CRAC channels 3,4 . This protein contains four transmembrane domains, is located in the plasma membrane and, therefore, may represent the CRAC channel itself, a subunit of the channel, or a regulatory molecule that couples to the channel. When overexpressed individually, neither STIM1 nor CRACM1 can significantly potentiate I CRAC 1-4 .To address the potential interaction of STIM1 and CRACM1, both proteins were overexpressed individually, or in combination, in HEK293 and Jurkat T cells and the CRAC (Fig. 1b, d). Consistent with previous work 1,9 , overexpression of STIM1 alone caused a small-to-modest increase in I CRAC in HEK293 and Jurkat cells (Fig. 1a, c). CRACM1 overexpression alone did not affect the CRAC currents induced by store depletion in HEK293 cells (Fig. 1a, b) and caused a small reduction in I CRAC in Jurkat cells (Fig. 1c, d). Unless simply due to a general effect of transfection or variability of I CRAC across preparations, this reduction may be due to some kind of dominant-negative effect. Taken together, the available data on CRACM1 and STIM1 suggest that the individually expressed proteins, although essential for I CRAC manifestation, cannot significantly amplify the current. This would indicate that these proteins are either not sufficient to generate large CRAC currents or that they are stoichiometrically linked and limit each others' ability to generate CRAC currents above normal. Therefore, we co-overexpressed both proteins in HEK293 cells (see Supplementary Information, Fig. S1) and assessed store-operated currents by patch clamp. HHS Public AccessThe co-overexpression of STIM1 and CRACM1, in both HEK293 and Jurkat cells, is sufficient to generate enormous membrane currents ...

Calcium dependence of T cell proliferation following focal stimulation

“…Using elegant screening approaches including positional cloning from the SCID patients, Feske and colleagues [14] have shown that a member of a new class of proteins, called ORAI (keepers of gates of heaven, from Greek mythology), is part of the CRAC channel complex. A similar conclusion was independently reached by others [15][16][17][18], who in addition showed that ORAI1 increased CRAC currents if expressed with the CRAC activator STIM1. STIM1 has been previously shown to mediate CRAC activation after Ca 2+ store depletion [19][20][21][22].…”

“…While the importance of Ca 2+ entry via STIM/ORAI/ CRAC for T cell proliferation, activation and even tolerance is undisputed [10,[13][14][15]23], very little is known about the correlation between T cell activation following formation of the IS and the amplitude and kinetics of the intracellular Ca 2+ concentration. This is surprising given the potential to control the adaptive immune response through changes in free intracellular calcium concentration ([Ca 2+ ] i ) during T cell activation.…”

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

“…First, we compared the fluorescence recovery after photobleaching (FRAP) of STIM1-CFP in unstimulated cells resting on a polylysine surface to that of STIM1-CFP in caps. We studied cells cotransfected with STIM1-CFP and untagged Orai1, because cotransfection of these two proteins is required for increased CRAC channel function (Mercer et al, 2006;Peinelt et al, 2006;Soboloff et al, 2006;Zhang et al, 2006). There was clear recovery of fluorescence from STIM1-CFP in the ER of unstimulated cells resting on polylysine, but little recovery of STIM1-CFP fluorescence when the bleached region was part of a cap structure in stimulated cells (Figure 3, A and C).…”

“…Orai1 is a four-pass integral membrane protein found in the PM that forms the channel. Overexpression of STIM1 with Orai1 results in very large CRAClike currents in Drosophila cell lines, Jurkat T-cells, RBL cells, and HEK293 cells (Mercer et al, 2006;Peinelt et al, 2006;Soboloff et al, 2006;Zhang et al, 2006). Recent studies have shown that Orai1 forms multimers and that targeted mutations in Orai1 alter the conductance properties of the CRAC channel Vig et al, 2006a;Yeromin et al, 2006;Gwack et al, 2007b).…”

Attenuated mesangial cell proliferation related to store-operated Ca2+ entry in aged rat: the role of STIM 1 and Orai 1

“…Orai 1 is a four-transmembranedomain protein. Knockdown of Orai 1 notably decreased SOCE (Peinelt et al 2006;Soboloff et al 2006). Indeed, it is widely accepted that Orai 1 and STIM 1 concertedly determine SOCE in a variety of cells.…”