Tarantula Toxin is Used to Report on Electrical Activity in Live Cells

Crucial experiments to develop a novel probe of cellular electrical activity were conducted in the Neurobiology course at the Marine Biological Laboratory (MBL) in 2013. Today, that optical probe, which combines a tarantula toxin with a fluorescent compound, is introduced in a paper in the Proceedings of the National Academy of Sciences.

The lead authors of the paper are Drew C. Tilley of University of California-Davis and the late Kenneth Eum, who was a teaching assistant in the Neurobiology course and a Ph.D. candidate at UC-Davis.

The probe takes advantage of the potent ability of tarantula toxin to bind to electrically active cells, such as neurons, while the cells are in a resting state. The team discovered that a trace amount of toxin combined with a fluorescent compound would bind to a specific subset of voltage-activated proteins (Kv2-type potassium ion channels) in live cells. The probe lights up cell surfaces with this ion channel, and the fluorescent signal dims when the channel is activated by electrical signals.

This is the first time that researchers have been able to visually observe these ion channels “turning on” without first genetically modifying them. All that is required is a means to detect probe location, suggesting that related probes could potentially one day be used to map neural activity in the human brain.

“This is a demonstration, a prototype probe. But the promise is that we could use it to measure the activity state of the electrical system in an organism that has not been genetically compromised,” says senior author Jon Sack, an assistant professor in the departments of Physiology and Membrane Biology at UC-Davis. Sack is a faculty member in the MBL Neurobiology course.

Since the probe binds selectively to one of the many different kinds of ion channels, it can help scientists disentangle the function of those specific channels in neuronal signaling. This can, in turn, lead to the identification of drug targets for neurological diseases and disorders.

“We have an incredible diversity of ion channels, and even of voltage-activated ion channels. The real trouble has been determining which ones perform which roles. Which ones turn on and when in normal nervous system functioning? Which are involved in abnormal states or syndromes?” Sack says. “The dream is to be able to see what the different types of ion channels are doing and when, to understand what they contribute to physiology and pathophysiology.”

These probes respond to movement of ion channel voltage sensors, and it is particularly fulfilling to have conducted some of this work at the MBL, Sack says. The first measurements of voltage sensor movement were conducted at the MBL in the early 1970s by Clay M. Armstrong and Francisco Bezanilla (Nature 242: 459-461, 1973). Armstrong and Bezanilla used electrophysiological methods to measure the movement of voltage sensors. The spider toxin probe creates an optical signal when voltage sensors move, and no electrophysiology or genetic mutation of the channels is required.

Hunger Games: How the brain ‘browns’ fat to aid weight loss

Researchers at Yale School of Medicine have uncovered a molecular process in the brain known to control eating that transforms white fat into brown fat. This process impacts how much energy we burn and how much weight we can lose. The results are published in the Oct. 9 issue of the journal Cell.

Obesity is a rising global epidemic. Excess fatty tissue is a major risk factor for type 2 diabetes, cardiovascular disease, hypertension, neurological disorders, and cancer. People become overweight and obese when energy intake exceeds energy expenditure, and excess calories are stored in the adipose tissues. The adipose organ is made up of both white and brown fat. While white fat primarily stores energy as triglycerides, brown fat dissipates chemical energy as heat. The more brown fat you have, the more weight you can lose.

It has previously been shown that energy-storing white fat has the capacity to transform into energy-burning “brown-like” fat. In this new study, researchers from the Yale Program in Integrative Cell Signaling and Neurobiology of Metabolism, demonstrate that neurons controlling hunger and appetite in the brain control the “browning” of white fat.

Lead author Xiaoyong Yang, associate professor of comparative medicine and physiology at Yale School of Medicine, conducted the study with Tamas Horvath, professor and chair of comparative medicine, and professor of neurobiology and Obstetrics/gynecology at Yale School of Medicine, and their co-authors.

The team stimulated this browning process from the brain in mice and found that it protected the animals from becoming obese on a high-fat diet. The team then studied the molecular changes in hunger-promoting neurons in the hypothalamus and found that the attachment of a unique sugar called “O-GlcNAc” to potassium ion channels acts as a switch to control brain activity to burn fat.

“Our studies reveal white fat “browning” as a highly dynamic physiological process that the brain controls,” said Yang. “This work indicates that behavioral modifications promoted by the brain could influence how the amount of food we eat and store in fat is burned.”

Yang said hunger and cold exposure are two life-history variables during the development and evolution of mammals. “We observed that food deprivation dominates over cold exposure in neural control of white fat browning. This regulatory system may be evolutionarily important as it can reduce heat production to maintain energy balance when we are hungry. Modulating this brain-to-fat connection represents a potential novel strategy to combat obesity and associated illnesses.”

(Image caption: A consensus shape for the calcium ion channel in the worm’s pain receptor nerve that was reached by computer modeling. Credit: Damian van Rossum and Andriy Anishkin, Duke University)

Surprising New Role for Calcium in Sensing Pain

When you accidentally touch a hot oven, you rapidly pull your hand away. Although scientists know the basic neural circuits involved in sensing and responding to such painful stimuli, they are still sorting out the molecular players.

Duke researchers have made a surprising discovery about the role of a key molecule involved in pain in worms, and have built a structural model of the molecule. These discoveries, described Sept. 2 in Nature Communications, may help direct new strategies to treat pain in people.

In humans and other mammals, a family of molecules called TRP ion channels plays a crucial role in nerve cells that directly sense painful stimuli. Researchers are now blocking these channels in clinical trials to evaluate this as a possible treatment for various types of pain.

The roundworm Caenorhabditis elegans also expresses TRP channels — one of which is called OSM-9 — in its single head pain-sensing neuron (which is similar to the pain-sensing nerve cells for the human face). OSM-9 is not only vital for detecting danger signals in the tiny worms, but is also a functional match to TRPV4, a mammalian TRP channel involved in sensing pain.

In the new study, researchers created a series of genetic mutant worms in which parts of the OSM-9 channel were disabled or replaced and then tested the engineered worms’ reactions to overly salty solution, which is normally aversive and painful.

Specifically, the mutant worms had alterations in the pore of the OSM-9 channels in their pain-sensing neuron, which gets fired up upon channel activation to allow calcium and sodium to flow into the neuron. That, in turn, was thought to switch on the neural circuit that encodes rapid withdrawal behavior — like pulling the finger from the stove.

“People strongly believed that calcium entering the cell through the TRP channel is everything in terms of cellular activation,” said lead author Wolfgang Liedtke, M.D., Ph.D., an associate professor of neurology, anesthesiology and neurobiology at Duke University School of Medicine and an attending physician in the Duke Pain Clinics, where he sees patients with chronic head-neck and face-pain.

With then-graduate student Amanda Lindy, “we wanted to systemically mutagenize the OSM-9 pore and see what we could find in the live animal, in its pain behavior,” Liedtke said.

To the group’s surprise, changing various bits of OSM-9’s pore did not change most of the mutant worms’ reactions to the salty solution. However, these mutations did affect the flow of calcium into the cell. The disconnect they saw suggested the calcium was not playing a direct role in the worms’ avoidance of danger signals.

Calcium has been thought to be indispensable for pain behavior — not only in worms’ channels but in pain-related TRP channels in mammals. So results from the engineered OSM-9 mutant worms will change a central concept for the understanding of pain, Liedtke said.

To see whether calcium might instead play a role in the worms’ ability to adapt to repeated painful stimuli, the group then repeatedly exposed pore-mutant worms to the aversive and pain stimuli.

After the tenth trial, a normal worm becomes less sensitive to high salt. But one mutant worm with a minimal change to one specific part of its OSM-9 pore — altered so that calcium no longer entered but sodium did — was just as sensitive on the tenth trial as on the first.

The results confirmed that calcium flow through the channel makes the worms more adaptable to painful stimuli; it helps them cope with the onslaught by desensitizing them. This could well represent a survival advantage, Liedtke said.

To put the findings into a structural context, Liedtke collaborated with computational protein scientists Damian van Rossum and Andriy Anishkin from Penn State University, who built a structural model of OSM-9 that was based on established structures of several of the channel’s relatives, including the recently resolved structure of TRPV1, the molecule that senses pain caused by heat and hot chili peppers.

The team was then able to visualize the key parts of the OSM-9 pore in the context of the entire channel. They understood better how the pore holds its shape and allows sodium and calcium to pass.

Liedtke said that understanding this structure could be a great help in designing compounds that will not completely block the channel but will just prevent calcium from entering the cell. Although calcium helps desensitize worms to painful stimuli in the near term, it might set up chronic, pathological pain circuits in the long term, Liedtke said.

So, as a next step, the group plans to assess the longer-term effects calcium flow has in pain neurons. For example, calcium could change the expression of particular genes in the sensory neuron. And such gene expression changes could underlie chronic, pathologic pain.

“We assume, and so far the evidence is quite good, that chronic, pathological pain has to do with people’s genetic switches in their sensory system set in the wrong way, long term. That’s something our new worm model will now allow us to approach rationally by experimentation,” Liedtke said.

Bad learning

University of Iowa researchers have discovered a new form of neurotransmission that influences the long-lasting memory created by addictive drugs, like cocaine and opioids, and the subsequent craving for these drugs of abuse. Loss of this type of neurotransmission creates changes in brains cells that resemble the changes caused by drug addiction.

The findings, published June 22 in the journal Nature Neuroscience, suggest that targeting this type of neurotransmission might lead to new therapies for treating drug addiction.

“Molecular therapies for drug addiction are pretty much non-existent,” says Collin Kreple, UI graduate student and co-first author of the study. “I think this finding at least provides the possibility of a new molecular target.”

The new form of neurotransmission involves proteins called acid-sensing ion channels (ASICs), which have previously been shown to promote learning and memory, and which are abundant in a part of the brain that is involved in drug addiction. The researchers, led by John Wemmie, professor of psychiatry in the UI Carver College of Medicine, reasoned that disrupting ASIC activity in this brain region (the nucleus accumbens) should reduce learned addiction-related behaviors. However, their experiments showed that loss of ASIC signaling actually increases learned drug-seeking in mice.

When mice learned to associate one side of a chamber with receiving cocaine, animals that lacked the ASIC protein developed an even stronger preference for the “cocaine side” than control mice, suggesting that loss of ASIC had increased addiction behavior. The same result was seen for morphine, another drug of abuse, which has a different mechanism of action than cocaine.

"Always before, the data suggested that when you get rid of ASICs, learning and memory are impaired," Wemmie says. "So we expected the same trend when we studied reward-related learning and behavior and we were surprised to find the opposite."

In a second experiment, rats learned to press a lever to self-administer cocaine. Blocking or removing ASIC in the rat brains caused the animals to self-administer more cocaine than control animals. Conversely, increasing the amount of ASIC by over-expressing the protein seemed to decrease the animals’ craving for cocaine.

"There are many forms of addiction," says Wemmie, who also holds appointments in the UI Departments of Molecular Physiology and Biophysics and Neurosurgery, and with the Iowa City VA Medical Center. "We’d like to see if these mechanisms also apply to other addictions besides cocaine and morphine. And, we want to move forward to see if this pathway can be used to target addiction."

Novel neurotransmission

As the name suggests, acid-sensing ion channels are activated by acid, in the form of protons. This research and a second UI study recently published in PNAS show that protons and ASICs form a previously unrecognized neurotransmitter pair that helps neurons communicate in a novel way; and appear to influence several forms of learning and memory, including fear, as well as addiction.

Manipulating the activity of ASICs or the level of protons (acidity) may provide a new way to treat addiction.

"We are still a long way from using these findings to create a therapy," notes Yuan Lu, co-first author and UI postdoctoral scholar. "The key significance of this study is that we have found new, different targets [that might allow us to inhibit the addiction behavior].”

Drugs change the brain

Previous research has shown that drug abuse and addiction physically alter the connections between neurons (synapses) that are important for the creation and storage of memories. Although normal learning requires synapses to be dynamic and plastic, exposure to addictive drugs abnormally increases synaptic plasticity in a way that is thought to underlie drug-related learning and addiction behaviors. The UI study found that absence of ASIC-proton mediated neurotransmission also increased synaptic plasticity in a way that resembled the changes created by addiction and drug withdrawal.

"It seemed like everything we looked at (physiology and structural changes) really paralleled what you would see in an animal undergoing drug withdrawal, even though these animals missing ASIC had never been exposed to drugs," Kreple says.

Overall the study findings suggest that ASIC-related neurotransmission in the nucleus accumbens may play a role in reducing synaptic plasticity and appropriately stabilizing synapses.

Neuroscience’s grand question

When your car needs a new spark plug, you take it to a shop where it sits, out of commission, until the repair is finished. But what if your car could replace its own spark plug while speeding down the Mass Pike? 

Of course, cars can’t do that, but our nervous system does the equivalent, rebuilding itself continually while maintaining full function. 

Neurons live for many years but their components, the proteins and molecules that make up the cell, are continually being replaced. How this continuous rebuilding takes place without affecting our ability to think, remember, learn or otherwise experience the world is one of neuroscience’s biggest questions.

And it’s one that has long intrigued Eve Marder, the Victor and Gwendolyn Beinfield Professor of Neuroscience. As reported in Neuron on May 21, Marder’s lab has built a new theoretical model to understand how cells monitor and self-regulate their properties in the face of continual turnover of cellular components.

Ion channels, the molecular gates on the surface of cells, determine neuronal properties needed to regulate everything from the size and speed of limb movement to how sensory information is processed. Different combinations of types of ion channels are found in each kind of neuron. Receptors are the molecular ‘microphones’ that enable neurons to communicate with each other.

Receptors and ion channels are constantly turning over, so cells need to regulate the rate at which they are replaced in a way that avoids disrupting normal nervous system function. Scientists have considered the idea of a ‘factory’ or ‘default’ setting for the numbers of ion channels and receptors in each neuron. But this idea seems implausible because there is so much change in a neuron’s environment over the course of its life. 

If there is no factory setting, then neurons need an internal gauge to monitor electrical activity and adjust ion channel expression accordingly, the team asserts. Because a single neuron is always part of a larger circuit, it also needs to do this while maintaining homeostasis across the nervous system.

The Marder lab built a new theoretical model of ion channel regulation based on the concept of an internal monitoring system. The team, comprised of postdoctoral fellow Timothy O’Leary, lab technician Alex Williams, Alessio Franci, of the University of Liege in Belgium, and Marder, discovered that cells don’t need to measure every detail of activity to keep the system functioning. In fact, too much detail can derail the process. 

“Certain target properties can contradict each other,” O’Leary says. “You would not set your air conditioning to 64 degrees and your heat to 77 degrees. One might win over the other but they would be continually fighting each other and you would end up paying a big energy bill.”

The team also learned that cells can have similar properties but different ion channel expression rates — like cellular homophones, they sound alike but look very different. 

The model showed that the very internal monitoring system designed to control runaway electrical activity can actually lead to neuronal hyperexcitability, the basis of seizures. Even if set points are maintained in single neurons, overall homeostasis in the system can be lost. 

The study represents an important advance in understanding the most complex machinery ever built — the human brain. And it may lead to entirely different therapeutic strategies for treating diseases, O’Leary says. “To understand and cure some diseases, we need to pick apart and understand how biological systems control their internal properties when they are in a normal healthy state, and this model could help researchers do that.”

(Image caption: This ribbon diagram shows three ankyrin repeats, a common structure found in receptor proteins that sense either cold or hot temperatures. A Duke team has identified three single-point mutations that can invert temperature-sensitivity, turning a cold-sensor into a heat-sensor. All three of these mutations are located in on a single ankyrin repeat. Credit: Grandl Lab, Duke University)

Small Mutation Changes Brain Freeze to Hot Foot

Ice cream lovers and hot tea drinkers with sensitive teeth could one day have a reason to celebrate a new finding from Duke University researchers. The scientists have found a very small change in a single protein that turns a cold-sensitive receptor into one that senses heat.

Understanding sensation and pain at this level could lead to more specific pain relievers that wouldn’t affect the central nervous system, likely producing less severe side effects than existing medications, said Jorg Grandl, Ph.D., an assistant professor of neurobiology in Duke’s School of Medicine who led the research team.

Temperature-induced pain, also called thermal pain, occurs when the body’s sensory neurons come in contact with temperatures above or below a certain threshold, such as plunging a limb into freezing water.

"We want to understand how either hot or cold temperatures can activate the sensors of hot and cold temperatures in the body," Grandl said.

Previous research has identified transient receptor potential (TRP) ion channels as being highly sensitive to either cold or hot temperatures. TRP ion channels are porous proteins that play a role in initiating electrical signals by controlling the flow of charged ions across the cell membrane.

It’s still unclear how temperatures make this happen, but the Grandl team’s research reveals that single-letter changes in DNA, called point mutations, are sufficient to make cold-sensitive TRP ion channels become sensitive to hot temperatures instead.

"There is strong interest in understanding temperature-sensitive molecules from a functional perspective because they are promising targets for developing analgesic compounds to treat chronic pain," said Grandl, who is also a member of the Duke Institute for Brain Sciences. "It is something we currently do not treat well. So, one promising strategy is to stop pain where it is initially sensed — at that first molecule that functions as a sensor of pain."

In a study appearing online early May 8 in the journal Neuron, Grandl’s team focused on TRPA1, an ion channel best known as a sensor for pain caused by environmental irritants and pungent chemicals, such as mustard oil, the active compound found in wasabi.

Grandl’s colleagues, postdoctoral fellow Sairam Jabba and research technician Raman Goyal, investigated whether single-point mutations could change cold-activated mouse TRPA1 into heat-activated. They formed this hypothesis because, in some other animals, including Drosophila fruit-flies and rattlesnakes, TRPA1 is naturally heat-activated.

To identify these structures, the team created a library of 12,000 mutant clones of the cold-activated mouse TRPA1 ion channel and randomly inserted one or two point mutations into each clone. After placing single clones into the individual slots of a 384-well plate and heating it from 25 degrees Celsius to 45 C in a matter of seconds, they were able to measure the thermal sensitivity of each mutant protein.

This screening pinpointed seven clones that showed strong activation when exposed to heat. Gene sequencing of these clones revealed 12 mutations that could potentially be responsible for changing the mouse TRPA1 from cold-activated to heat-activated. Out of these 12 mutations, Jabba and Goyal identified three mutations powerful enough to individually make that switch in TRPA1.

The mutations all turned out to be located within a single small domain of the ion channel protein known as ankyrin repeat six, indicating this domain plays a role in determining cold or heat activation. Ankyrin repeats are often responsible for managing protein-to-protein interactions, but their precise function in TRPA1 had not been previously known.

Interestingly, these single-point mutations didn’t change the ion channels’ responses to chemicals, such as mustard oil.

"This was very surprising and it demonstrates that making a single-point mutation produced a profound change in the temperature sensitivity of the protein, but it did not affect the chemical sensitivity," Grandl said. "It shows these mechanisms are to some degree distinct."

Grandl said that taken together, the findings also suggest that the effectiveness of such a small mutation might have been key to a single ancestral ion channel evolving into the wide diversity of temperature-activated ion channels we see today.

(Image caption: Channelrhodopsins before (upper left) and after (lower right) molecular engineering, shown superimposed over an image of a mammalian neuron. In the upper left opsin, the red color shows negative charges spanning the opsin that facilitated the flow of positive (stimulatory) ions through the channel into neurons. In the newly engineered channels (lower right), those negative charges have been changed to positive (blue), allowing the negatively charged inhibitory chloride ions to flow through. Credit: Andre Berndt, Soo Yeun Lee, Charu Ramakrishnan, and Karl Deisseroth.)

Researchers Build New “Off Switch” to Shut Down Neural Activity

Nearly a decade ago, the era of optogenetics was ushered in with the development of channelrhodopsins, light-activated ion channels that can, with the flick of a switch, instantaneously turn on neurons in which they are genetically expressed. What has lagged behind, however, is the ability to use light to inactivate neurons with an equal level of reliability and efficiency. Now, Howard Hughes Medical Institute (HHMI) scientists have used an analysis of channelrhodopsin’s molecular structure to guide a series of genetic mutations to the ion channel that grant the power to silence neurons with an unprecedented level of control.

The new structurally engineered channel at last gives neuroscientists the tools to both activate and inactivate neurons in deep brain structures using dim pulses of externally projected light. HHMI early career scientist Karl Deisseroth and his colleagues at Stanford University published their findings April 25, 2014 in the journal Science. “We’re excited about this increased light sensitivity of inhibition in part because we think it will greatly enhance work in large-brained organisms like rats and primates,” he says.

First discovered in unicellular green algae in 2002, channelrhodopsins function as photoreceptors that guide the microorganisms’ movements in response to light. In a landmark 2005 study, Deisseroth and his colleagues described a method for expressing the light-sensitive proteins in mouse neurons. By shining a pulse of blue light on those neurons, the researchers showed they could reliably induce the ion channel at channelrhodopsin’s core to open up, allowing positively charged ions to rush into the cell and trigger action potentials. Channelrhodopsins have since been used in hundreds of research projects investigating the neurobiology of everything from cell dynamics to cognitive functions.

A few years later came the deployment of halorhodopsins, light-sensitive proteins selective for the negatively charged ion chloride. These proteins, derived from halobacteria, provided researchers with a tool for the light-controlled inactivation of neurons. A major limitation of these proteins, however, is their inefficiency. Unlike channelrhodopsin, halorhodopsin is an ion pump, meaning that only one chloride ion moves across the neuron’s membrane per photon of light. “What that translates into is you get partial inhibition,” Deisseroth says. “You can inhibit neurons, but in the living animal it’s not always complete.”

Searches for a naturally occurring light-sensitive channel with a pore permeable to negatively charged ions have come up empty handed. “We searched,” Deisseroth says. “We did big genomic searches and found many interesting channelrhodopsins and lots of pumps, but we never found an inhibitory channel in nature.”

The team’s fruitless exploration led them to try modifying the molecular structure of channelrhodopsin so that its pore would shuttle negative ions into the cell. “To do that you need to know what the channel pore looks like at the angstrom level,” Deisseroth says. “What we really needed was the high-resolution crystal structure.” In 2012, working with a group in Japan, Deisseroth and his colleagues captured the structure of a chimera of channelrhodopsin called C1C2 using X-ray crystallography.

A molecular analysis of channelrhodopsin’s pore suggested that swapping out certain negatively charged amino acid residues lining the pore with positive residues could reverse the electrostatic potential of the channel, making it more conductive to negatively charged ions such as chloride. To achieve this molecular switcheroo, the researchers performed dozens of single site-directed mutations. Several mutations conferred selectivity for chloride, but the channels failed to conduct current. So, the team screened hundreds of combinations of mutations. “In a systematic process we found first a combination of four mutations, and then a group of five mutations, that seemed to change selectivity,” says Deisseroth. “We put those together into a nine-fold mutated channel and that one, amazingly, was chloride selective.”

Not only does the new channel—dubbed iC1C2 for “inhibitory C1C2”—allow the selective passage of chloride ions, it greatly reduces the likelihood of action potentials by making the neuron more “leaky,” a function not possible in ion pumps like halorhodopsin.

Deisseroth’s team made a final mutation to a cysteine residue in iC1C2 that makes the channel both bi-stable and orders of magnitude more sensitive to light. When activated by blue light, the mutated channels remain open for up to minutes at a time, while exposing the channels to red light makes them close quickly. This level of long-term control is useful in developmental studies where events play out over minutes to hours. The long channel open times also mean that neurons can essentially integrate chloride currents over longer time scales and, therefore, weaker light can be used to inhibit the neurons. Increased light sensitivity translates to less light-induced damage to neural tissue, the ability to reach deep brain structures, and the possibility of controlling brain functions that involve large regions of the brain.

“This is something we’ve sought for many years and it’s really the culmination of many streams of work in the lab—crystal structure work, mutational work, behavioral work —all of which have come together here,” Deisseroth says.

Team Solves Decades-Old Mystery of How Cells Keep from Bursting

A team led by scientists at The Scripps Research Institute (TSRI) has identified a long-sought protein that facilitates one of the most basic functions of cells: regulating their volume to keep from swelling excessively.

The identification of the protein, dubbed SWELL1, solves a decades-long mystery of cell biology and points to further discoveries about its roles in health and disease—including a serious immune deficiency that appears to result from its improper function.

“Knowing the identity of this protein and its gene opens up a broad new avenue of research,” said the study’s principal investigator Ardem Patapoutian, a Howard Hughes Medical Institute (HHMI) Investigator and professor at TSRI’s Dorris Neuroscience Center and Department of Molecular and Cellular Neuroscience.

The report appears as the cover story in the April 10, 2014 issue of the journal Cell.

Unraveling the Mystery

Water passes through the membrane of most cells with relative ease and tends to flow in a direction that evens out the concentration of dissolved molecules or “solutes.” “Water in effect follows the solutes,” explained Zhaozhu Qiu, a member of the Patapoutian laboratory who was first author of the study. “Any decrease in the solute concentration outside a cell or an increase within the cell will make the cell swell with water.”

For decades, experiments have demonstrated the existence of a key relief valve for this swelling: an unidentified ion channel in the cell membrane, dubbed VRAC (volume-regulated anion channel). VRAC opens in response to cell swelling and permits an outflow of chloride ions and some other negatively charged molecules—which water molecules follow, thus reducing the swelling.

“For the past 30 years, scientists have known that there is this VRAC channel, and yet they haven’t known its molecular identity,” said Patapoutian.

Finding the proteins that make VRAC and their genes was a goal that had eluded prior attempts because of the technical hurdles involved. However, in the new study, Qiu and his colleagues were able to set up a rapid, “high-throughput” screening test based on fluorescence. They engineered human cells to produce a fluorescent protein whose glow would be quenched when the cells became swollen and VRAC channels opened.

With the help of automated screening specialists at the La Jolla-based Genomics Institute of the Novartis Research Foundation (GNF), which recently began a broad new collaboration agreement with TSRI, the team cultured large arrays of the cells and, using a technique known as RNA interference, blocked the activity of a different gene for each clump of cells.

The idea was to watch for the groups of cells that continued to glow—indicating that the gene inactivation had disrupted VRAC.

In this way, with several rounds of tests, the team sifted through the human genome and ultimately found one gene whose disruption reliably terminated VRAC activity. It was a gene that had been discovered in 2003 and catalogued as “LRRC8.” Although it appeared to code for a cell-membrane-spanning protein—as one would expect for an ion channel—almost nothing else was known about it.

The team renamed it SWELL1.

Potential Roles in Disease

Investigating further, the researchers showed that SWELL1 does indeed localize to the cell membrane as an ion channel protein would. Experiments by Adrienne Dubin, a staff scientist at TSRI, showed that certain mutations of SWELL1 alter the VRAC channel’s ion-passing properties—indicating that SWELL1 is a central feature of the ion channel itself.

“It is at least a major part of the VRAC channel for which cell biologists have been searching all this time,” said Patapoutian.

Patapoutian, Qiu and their colleagues now will study SWELL1 further, including an examination of what happens to lab mice that lack the protein in various cell types.

Curiously, the gene for SWELL1 was first noted by scientists because a mutant, dysfunctional form of it causes a very rare type of agammaglobulinemia—a lack of antibody-producing B cells, which leaves a person unusually vulnerable to infections. That suggests that SWELL1 is somehow required for normal B-cell development.

“There also have been suggestions from prior studies that this volume-sensitive ion channel is involved in stroke because of the brain-tissue swelling associated with stroke and that it may be involved as well in the secretion of insulin by pancreatic cells,” said Patapoutian. “So there are lots of hints out there about its relevance to disease—we just have to go and figure it all out now.”