STIMulating Discoveries

Two independent labs identified STIM as the protein that senses depleted calcium stores in the endoplasmic reticulum and activates the channels through which calcium enters cells.

ILLUSTRATION: THOM GRAVES

Keeping calcium levels high in the endoplasmic reticulum is critical for protein synthesis and folding, and cellular functions such as smooth muscle contraction and T lymphocyte differentiation and proliferation depend upon calcium entry. Most cells utilize a pathway called store-operated calcium entry, whereby the emptying of intracellular calcium stores opens calcium release-activated calcium (CRAC) channels in the plasma membrane, allowing the influx of extracellular calcium to replenish stores in the endoplasmic reticulum.

Some aspects of the pathway have been characterized since the phenomenon was discovered in 1986, but for almost 20 years, the molecular messengers translating calcium-store depletion to CRAC-channel activation remained shrouded in mystery. Then, in 2005, an essential part of the mystery was solved.

That year, three papers, two of which are Hot Papers, detailed the discovery of a key signaling protein: stromal interaction molecule (STIM). The first of these three studies to be published was conducted by Jack Roos and Kenneth Stauderman, then with Torrey Pines Therapeutics, a California pharmaceutical company, and a team from Michael Cahalan's lab at University of California, Irvine. Using RNAi, they screened 170 genes in Drosophila S2 cells and found that knocking down the gene for STIM essentially halted store-operated calcium entry. ¹ They also found that the suppression of one of the two mammalian homologues of Drosophila STIM, called STIM1, inhibited store-operated calcium influx in three different human cell lines.

"The minute I first saw the data on STIM, I knew right away it was correct," says James Putney, head of the National Institutes of Health's calcium regulation section (who was not involved in any of the papers). "It was just like a dam breaking. We had a physiological phenomenology, but we had no proteins, no molecules. It really opened the door to move the field forward."

Two months later, Tobias Meyer's Stanford University lab published a paper that confirmed STIM1's calcium store depletion sensor role in HeLa cells and went further to suggest that STIM2 (the other mammalian STIM homologue) was playing a similar role. ² The Stanford group also observed STIM1 rapidly translocating from the ER to points near the plasma membrane in response to calcium-store depletion, suggesting a mechanism for the protein's regulation of the store-operated calcium pathway. They also showed that STIM1's calcium-binding domain was likely playing a role in this interaction with the yet uncharacterized CRAC channel proteins. Then, in the autumn of 2005, Cahalan's group (along with Roos and Stauderman) published similar results, reporting that STIM1 was acting as the calcium sensor and that it migrated to the plasma membrane to activate CRAC channels. ³

A key difference between the Meyer and Cahalan papers was that the latter proposed a mechanistic model in which STIM1 physically inserted into the plasma membrane. Researchers have since shown, using cells transfected with STIM1 mutants incapable of inserting into the plasma membrane, that the protein is still able to initiate calcium influx through CRAC channels. ⁴ "Our evidence was in favor of [STIM1 insertion into the plasma membrane]," says Cahalan. "It turns out that that insertion may not be required for acute channel activation."

Recently, other researchers conducted genome-wide RNAi screens on Drosophila to uncover another key component to store-operated calcium entry: the putative CRAC channel protein itself, olf186-F (Orai), which established an entirely new family of channels. Last year three separate labs identified Orai as the pore-forming subunit that interacts with STIM to incite store-operated calcium influx. The first of these to be published (also a Hot Paper) identified a genetic defect in Orai1 - the human Orai homologue - as responsible for the loss of CRAC channel function and subsequent immunodeficiency in patients with severe combined immune deficiency. ⁵

With STIM and Orai identified as the essential components of store-operated calcium entry, the calcium signaling community has taken to characterizing the molecular interaction between the two. "We still don't know how STIM talks to Orai," says Putney, "That's still up in the air to some degree."

Though the nature of STIM and Orai's molecular interaction has yet to be elucidated, Patrick Hogan, of the Immune Disease Institute at Harvard and a collaborator on the study that originally identified Orai, says that the discoveries of the two proteins united the calcium-signaling community. "Once STIM and Orai came out, this whole fractious field that has had trouble agreeing on anything for 15 years or more, came to the conclusion that they really were important players in [store-operated calcium entry]," he says.