Calcium Signaling Out of the Gate

Merged images of cultured cells expressing STIM1 (red) and Orai1 (green), taken before (top) and after (bottom) calcium store depletion.

Courtesy of Paul Hoover and Richard Lewis / Stanford University

In one of the cell's key controls of internal calcium levels, channels at the cell membrane open in response to changes in calcium levels in the endoplasmic reticulum (ER). These plasma membrane channels, called calcium-release activated calcium (CRAC) channels, were first identified electrophysiologically about 20 years ago. Because their tiny conductance (about 100 times lower than that of any other known calcium channel) makes individual channel currents very difficult to detect, however, both the molecular mechanism of this signaling pathway and the identity of the channel, have until recently remained a mystery.

Just two years ago, shortly after the discovery of a protein called stromal interacting molecule (STIM), which acts as the ER calcium sensor that activates CRAC channels when calcium levels drop, researchers began to home in on the identity of the channel itself. (See B. Grant, "STIMulating Discoveries," The Scientist, October 2007.)

In 2006, three studies using genome-wide Drosophila screens all converged on a novel protein, Orai, as an essential component of CRAC channel function; when overexpressed with STIM, the CRAC current surged. The first study, a Hot Paper this month, was led by Anjana Rao at Harvard Medical School. It hinted at the CRAC channel's physiological role: Cells taken from two patients with a rare form of severe combined immune deficiency (SCID) had a mutation in Orai that almost completely slashed the CRAC current. ¹

A few months later, another trio of publications conclusively identified Orai as the channel's critical pore-forming element by showing that point mutations in the protein's predicted transmembrane domains changed the channel's ion selectivity and conductance properties. The results were "the icing on the cake," says Michael Cahalan at University of California, Irvine, the main author of one of the studies, which is also a Hot Paper this month. ² Nailing down the identity of both the sensor and the channel itself, he says, has created "a tremendous surge of excitement in the calcium signaling field" and "triggered a blur of publications."

Even with the identity of the channel in hand, however, many elements of the unfolding CRAC channel story remain controversial - down to the channel's name. According to Reinhold Penner of the University of Hawaii, "Orai" - named after three Greek goddesses who are the keepers of the gates of heaven - is a gross misnomer. "STIM is the keeper," he says, while the channel "is the gate."

After the discovery of Orai, researchers wondered about how the sensor and the channel communicate with each other. Upon calcium store depletion, STIM proteins reorganize from their diffuse positions throughout the ER membrane to form discrete punta close to the plasma membrane. Richard Lewis' group at the Stanford University School of Medicine showed that activated Orai colocalizes with STIM, concentrating calcium influx to the membrane region containing both proteins. ³ STIM and Orai also interact in energy-transfer imaging experiments, which is probably the best indication so far that the two act in complex.

Still, other views are out there. One lab suggests that a second messenger delivers the signal from STIM to Orai. Other researchers implicate a different group of proteins as a key element of the CRAC signaling pathway, and possibly even as the CRAC channel itself (instead of Orai). The results of those studies, however, remain "a minority view," says Lewis.

Several parts of the molecular picture are still missing, starting with a clear understanding of Orai's stoichiometry. The only clues so far, from Trevor Shuttleworth's group at the University of Rochester, suggest that the channel is a tetramer. ⁴ Cahalan's group, however, has come up with a different model (currently in press). Researchers are also dissecting the contributions of each of the three mammalian Orai variants; studies so far suggest that Orai1 and Orai2 have similar properties and distribution, with Orai3 varying more dramatically. Because the channel is so difficult to detect, most CRAC channel studies so far have overexpressed Orai and STIM.

What's clear, researchers agree, is that the channel is very strange, with no homology to any previously described ion channels. Still, says Lewis, progress in just two years has been tremendous.

Knockout studies are exploring the channel's cellular function, in particular with mouse knockouts for the two STIM variants and the three Orai variants found in mammals. In February 2008, Jean-Pierre Kinet's group at Harvard published the first results of an Orai1 knockout. ⁵ The mice showed deficits in mast cells but not T cells, which is surprising in light of the Orai1 mutations observed in the SCID patients, says Stefan Feske from New York University, as well as the fact that Orai1 is the primary variant in human T cells. Some researchers argue that Orai2 and Orai3 may compensate for loss of Orai1 function, but Kinet's technique might also have incompletely knocked out the gene.

Meanwhile, researchers say pharmaceutical companies have begun to investigate the CRAC channel blockers to reduce the toxicity of immunosuppressant drugs such as cyclosporine A, used to prevent host rejection after organ transplantation. "We and others think there's a real opportunity for immunosuppression to be mediated by Orai1," says Cahalan.