The molecular identity and precise function of a commonly expressed cell-membrane chloride channel has long been a mystery. A recent study in Science uncovers the gene responsible for the channel, potentially shedding light on its role in the body. Researchers dubbed the gene PAC for proton activated chloride channel. “Now that there’s a genetic handle on this ion channel,” says research and clinical neuroscientist John Wemmie at the University of Iowa in Iowa City, “it’s sort of off to the races.”
Two decades ago, another group of ion channels had a similar story, he says. They were believed to play a role in the pain caused by acidifying tissue in inflammatory conditions. Beyond that, they weren’t well understood. Then, in 1997, research in Nature revealed the molecular identity of the protein family, called ASICs, for acid-sensing ion channels, responsible for a current of positively charged ions, in response to acid.
At the time, neuroscientists knew some general effects of acidic pH, Wemmie explains. Acidity in the brain was historically associated with disease. The constricted blood flow of a stroke, for example, acidifies tissue.
In the last 20 years, research findings have suggested that pH fluctuation can accompany normal brain function. This has implications for everything from stroke, epilepsy, and migraines, to learning and memory, Wemmie adds. But “we still don’t have a great grasp of both the causes and the consequences of these changes in pH,” he says. New discoveries, including the recent findings, could help fill out the picture.
Before the new work in Science, researchers knew that the proton chloride channel activated around pH 5.5 at room temperature, which allows chloride, the most abundant free anion in animal cells, to flow down its electrochemical gradient, explains Zhaozhu Qiu, a molecular biologist at Johns Hopkins University School of Medicine in Baltimore Maryland, and the principal investigator of the new study. Under the right conditions, chloride flows down its electrochemical gradient into a cell when the channel opens, allowing water to rush in too, which can cause cell swelling and even cell death. That could be one mechanism of tissue damage in diseases such as heart attack and stroke, he says, in which constricted blood supply leads to acidification.
But the gene for the ion channel was unknown. Qiu’s team identified it from a pool of more than 2,700 candidate genes by reducing their expression one-by-one in human embryonic kidney cells engineered to fluoresce. Cells with a functional chloride channel gene glowed until they were exposed to acidic conditions, when the pores opened and anion flow extinguished the cell’s light. When the researchers reduced expression of the right gene, the engineered cells kept glowing in acidic conditions, suggesting the chloride channel’s expression had decreased. Previously known by the generic name TMEM206, Qiu’s team labeled it PAC.
Once they had their candidate, the researchers used CRISPR to knock out the gene in several kinds of cells, then looked for an electrophysiological current indicative of the flow of chloride ions through the pore; they didn’t find it. “Lo and behold,” Qiu says, “knocking out this gene abolished the PAC current.” This confirmed it was the blueprint for the proton-activated chloride channel.
From there, the researchers re-introduced PAC to cells where the gene had been knocked out and confirmed the PAC current could be rescued. They also proved that PAC constitutes the membrane pore and is not an accessory subunit.
Zebrafish also express the PAC protein, suggesting these acid-activated channels are conserved in vertebrates, explains chemist Andrea Cavalli of the Italian Institute of Technology in Genoa, Italy. “It’s not limited to mammalian cells,” he says.
Hence, the protein may have a basic physiological role. One possibility, Qiu says, is that it helps waste processing organelles, such as lysosomes, regulate their pH while breaking down cellular garbage.
Qiu’s team also knocked out PAC in mice, confirming that the gene drives acid sensitive chloride channel activity in an animal model. They found that the neurons from the knockout mice were more resistant to acid-induced cell death; and the mice were partially-protected from brain damage after stroke. That suggests PAC does influence disease, though the precise mechanism remains unknown. Qiu says it could be a future target for drug development.
Discoveries of new ion channel genes are rare. Wemmie’s biggest issue with the paper? Waiting for more research to come out. “You’re left being tantalized,” he says, because the connections to disease are there, but the mechanisms are still opaque. “You’re just sort of left to wonder, ‘how is that happening?’”