Discovery of Calcium Channel Protein Illuminates T Cell Signaling

Mutant Molecule Linked to Severe Combined Immunodeficiency

A family’s rare genetic defect has helped researchers identify a key signaling component in T cells. The newly identified protein, Orai1, may be a piece of a long-sought calcium channel in T cells that is critical for lymphocyte function. When two siblings inherited two copies of a mutant form of Orai1, it caused a severe impairment of their immune systems. Now, more than a decade after their case was reported, Stefan Feske and Yousang Gwack in Anjana Rao’s lab have found the protein responsible for the disease. The research appears in the April 2 Nature.

Photo by Graham Ramsay

Taking two approaches, one an RNAi screen in fruit flies and the other a linkage analysis in a human family, Yousang Gwack (left) and Stefan Feske uncovered a gene necessary for proper calcium signaling in T cells.

Several years ago, Feske, one of the study’s two co–first authors, began investigating the case of two brothers with severe combined immunodeficiency (SCID) syndrome who were patients at the children’s hospital at the University of Freiburg, Germany, where he was a student. SCID is characterized by a severely incapacitated immune system and can arise from several genetic defects that interfere with the development and function of T cells. Feske’s preliminary studies suggested that the T cells of the two patients had impaired functioning of NFAT, a transcription factor that helps regulate critical lymphocyte genes.

The NFAT connection brought him to Rao, HMS professor of pathology at the CBR Institute for Biomedical Research, whose lab had first cloned the transcription factor. But Feske, now an assistant professor of pediatrics at the CBR, found that none of the four NFAT genes were mutated in the patients’ T cells, leading the team to look further upstream at proteins involved in regulating NFAT. “We walked our way up the signal transduction cascade,” Feske explained.

Dual Screens
NFAT is regulated by calcium signals in the cell. When receptors at the cell surface are stimulated, calcium reserves are released. When those are depleted, an unknown signal prompts the opening of calcium release–activated calcium (CRAC) channels at the membrane, allowing even more calcium in from the outside. This sustained influx activates NFAT molecules drifting in the cytoplasm, which travel to the nucleus to turn on gene transcription.

The team attacked the problem using two unbiased genetic approaches. Feske began with a genomewide screen of single nucleotide polymorphisms (SNPs) from the patients’ family. Realizing that it might be a challenge to find the gene just with human data, Rao also recruited co–first author Yousang Gwack to the laboratory to conduct an RNAi screen in fruit flies for regulators of NFAT.

The parents of the two children were first cousins, suggesting that the disease might be the result of a recessive allele carried by both parents and harmfully paired in the children. Neither the parents nor any of the children’s relatives showed signs of immune deficiency, but Feske obtained blood samples from the extended family and tested their T cells for a more subtle defect.

In fact, the parents and 13 relatives had impaired calcium signaling in T cells, even though they had no clinical disease. When the T cells were exposed to low extracellular calcium concentrations, the rate of calcium influx was 50 percent lower than in controls. This test identified family members who carried a single copy of the mutant gene, allowing them to construct a more complete genetic family tree.

With the added information from the extended family, Feske and Rao teamed up with Mark Daly, HMS lecturer on medicine at Massachusetts General Hospital and the Broad Institute, to perform a linkage analysis of the family. They used a SNP array to identify regions of DNA that were identical only in DNA of the two siblings. “The traditional analysis pointed out six regions of the genome that were likely to harbor the actual disease gene,” said Daly. To narrow the field further, the team treated the minor calcium defect in the extended family as if it were a disease caused by a dominant gene. This second analysis pointed to a single region of the genome—one of the six that the previous screen had identified. With both analyses combined, the likelihood that this region was the correct one was 500,000 to one.

Flying Finish
Although the region was tightly linked to the SCID disease, it was home to more than 75 genes, so finding the particular disease-related mutation could have been a huge challenge. But the fruit fly screen allowed the team to find their target.

Fruit flies lack NFAT, but they have a calcium channel similar to the CRAC channel. Gwack, a research associate in pathology at the CBR, expressed human NFAT with a fluorescent label attached in the fruit fly cells, and then incubated the cells with double-stranded RNAs complementary to each of the 21,000 fruit fly genes. He stimulated the cells and looked for genes whose depletion by RNAi kept NFAT from traveling to the nucleus. Two genes had a major effect. One of them, known as Stim, had previously been identified by other labs as being important for calcium influx. The other novel gene had three human homologues, one of which was found in the same chromosomal region identified in Feske and Daly’s screen. Gwack named the fruit fly protein Orai and its three human homologues Orai1, 2, and 3, after the three keepers of the gates of heaven in Greek mythology.

Image courtesy of Stefan Feske

Tracking a mutation. To uncover the disease-causing genetic region in two siblings with severe combined immunodeficiency (SCID), researchers used a laboratory test to identify heterozygous carriers in the family. They conducted a linkage analysis first using the immediate family of the patients (pedigree A). They then analyzed the extended family and treated the heterozygous phenotype as the disease (pedigree B), which helped to narrow the field to one region of the genome.

The team confirmed that the siblings carried a point mutation in Orai1, and reconstituting the defective T cells with wild-type Orai1 restored function. “The two genomewide screens—one in humans and one in flies—came together very beautifully,” said Rao.

The team believes that Orai1 is a major component or regulator of the CRAC channel. The channel’s properties have been studied electrophysiologically for 15 years, but its molecular identity is unknown. With a protein target now available, researchers can study the calcium influx pathway in more detail. “I think it will open up a new field,” said Rao. “It’s not just a current anymore; we will be able to understand biochemically and molecularly how all the components work.”

—Courtney Humphries

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