A sharp increase in the concentration of calcium ions in a cell is a key biological signal. Now a vital component of a major route by which calcium ions flow into cells has been identified.
Communication between and within cells is essential for the development and survival of any complex organism. Cells converse with each other mainly through a complement of chemical messengers, including neurotransmitters and hormones, that impinge on the cell surface, generating further signals (second messengers) within the cell that elicit the appropriate responses. Although several hundred hormones, neurotransmitters and other molecules can stimulate cells, the number of intracellular second-messenger systems they activate is remarkably small. Perhaps the most widespread and ubiquitous of the second messengers is the calcium ion. Two reports, one in this issue by Feske et al. (page 179)1 and one in Science by Vig et al.2, uncover an essential component that allows cells to regulate their intracellular Ca2+ concentration.
A sharp rise in intracellular Ca2+ concentration can activate a disparate range of responses in almost all cells in the animal kingdom. Such rises stimulate neurotransmitter release, muscle contraction, cell metabolism, cell growth and proliferation, as well as processes culminating in cell death. Cells can increase their intracellular Ca2+ concentration in two ways: by releasing Ca2+ from intracellular stores, or by allowing Ca2+ across the cell membrane. The Ca2+ stores have a limited capacity, so Ca2+ influx into the cell drives most of the cellular responses.
A primary route for Ca2+ influx is through ‘store-operated channels’ in the cell membrane3. The prototypic store-operated channel is the ubiquitous CRAC (for Ca2+ release-activated Ca2+) channel4,5. Entry by this route is activated by a fall in Ca2+ within the endoplasmic reticulum6 — the labyrinthine network of membranes used to transport various substances around the cell. The endoplasmic reticulum usually holds a considerable stock of Ca2+ ions, without which it cannot function. However, a fall in Ca2+ concentration here, usually in response to the second messenger inositol 1,4,5-trisphosphate, somehow translates into the opening of store-operated Ca2+ channels. The molecular basis of this route of entry remains one of the more enduring mysteries in cell biology.
Once opened, CRAC channels enable Ca2+ ions to enter the cell. But despite their biological and clinical importance, very little is known about how these channels are activated, let alone their molecular composition. In breakthrough papers, Feske et al.1 report that a protein they call Orai1 (encoded by human gene FLJ14466) is an essential component of the CRAC channel complex, and Vig et al.2 identify the same gene (although they call the encoded protein CRACM1) and reach the same conclusion.
The work of Feske et al.1 built on a significant finding made last year that a protein called stromal interaction molecule (STIM1) is required for the activation of store-operated Ca2+ influx7,8. STIM1 spans the endoplasmic reticular membrane and has a Ca2+-binding structural motif that may ‘sense’ the store's Ca2+ concentration. As the store loses Ca2+, STIM1, which is diffusely dispersed throughout the endoplasmic reticular membrane, becomes redistributed into discrete spots (puncta) in the cell periphery. Whether it remains just below the membrane or is inserted into it remains controversial.
However, although necessary, the presence of STIM1 is not enough to activate CRAC channels. Patients with severe combined immunodeficiency (SCID) have impaired CRAC channel activity in their T cells that renders these immune cells defective. But STIM1 in these cells is normal, nor does overexpression of STIM1 restore Ca2+ influx9. So a molecule downstream of STIM1 must be responsible for the SCID defect.
Feske et al.1 demonstrate convincingly that this molecule is Orai1. To start with, they genetically mapped the mutation underlying SCID to a region on human chromosome 12. Because store-operated Ca2+ influx occurs in a similar manner in fruitflies and mammals, and because the fruitfly is the more easily manipulated in genetic experiments, the authors conducted a thorough search of the fruitfly genome for genes involved in store-operated Ca2+ influx. They found that the fruitfly gene encoding the Orai protein is a key component of the process. Crucially, a human relative of this gene, encoding Orai1, mapped to the same region on chromosome 12 as that linked to the SCID mutation. Elegant confirmation that Orai1 is responsible for the CRAC channel defect seen in SCID was provided when Feske et al. overexpressed Orai1 in SCID T cells: the normal protein rescued store-operated Ca2+ influx and CRAC channel activity, but a mutated version did not.
A similar search of the fruitfly genome by Vig et al.2 identified the same gene as being central to CRAC channel activation. Reducing the expression of this gene abolished CRAC activity in three different cell types. So two independent groups have identified the same protein as being fundamental to the activation of this ubiquitous Ca2+ influx pathway.
What is the role of Orai1/CRACM1? It is a cell-membrane protein, and its constituent amino-acid sequence suggests that it has four membrane-spanning segments. Could it be the elusive CRAC channel? This is not clear yet. The amino-acid sequence does not contain the ring of negatively charged glutamate amino acids that is typical of the selectivity filter of Ca2+ channels, nor does it show an obvious pore structure to allow passage of the ions through the membrane. However, the CRAC channel pore has unusual ion-permeation properties suggesting that it may differ from other known pores. Indeed, it is not even clear whether the CRAC complex is a channel through which ions would flow passively when it is open, or a transporter that, directly or indirectly, uses energy to move ions across the membrane. The way in which mutations in Orai1 affect the selectivity of the CRAC channel may help resolve this issue. But even if Orai1/CRACM1 is not the entire CRAC channel, it could be an indispensable subunit of a multimeric channel complex, or it might function as a key component of the activation mechanism (Fig. 1).
There is burgeoning evidence that store-operated channels operate in non-immune cells such as endothelia, epithelia and smooth muscle. It is puzzling, therefore, that SCID patients lacking functional CRAC channels suffer only immune disorders. An intriguing possibility is that Orai1/CRACM1 is essential for functional CRAC channels but that two closely related human proteins (Orai2 and Orai3) help to form biophysically distinct store-operated channels in these other tissues.
The shroud of mystery surrounding store-operated Ca2+ channels is at last being lifted by the forensic precision of molecular genetics. I anticipate rapid progress towards the complete molecular definition of CRAC channels. This will improve the prospects for developing therapeutic agents aimed at combating the growing list of human diseases associated with aberrant store-operated calcium influx6.
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Nature Cell Biology (2006)