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This is the introduction to a talk I gave in Oslo last Friday, Sept. 12, 2014, at a conference on science communications. A video of the complete talk, including the more serious part, can be seen at this link. It’s the last two film segments on the page.

Thank you Unni for that very kind introduction. It was all true, even the parts that sounded like some weird movie that you would probably never recommend to your friends. My name is Russ Hodge and it’s a great pleasure to speak at this wonderful event. I’ve been to Oslo many times and always enjoy the trip to your beautiful city. I come here every year the first week of December to teach a course on presentations skills to molecular biologists. I don’t know how successful it is, but nobody has ever died during it, or even been seriously injured, and they keep inviting me back, so you may draw your own conclusions.

I couldn’t remember exactly how many times I have been to Oslo, so last night I sat down and tried to figure it out. I came the first time in 2007, and have come one time every year since, so I could apply this formula:

2014 –  2007 = 7

I use the same formula when somebody asks me how old I am. Here, of course, the calculation doesn’t work out; you have to add another trip to include both 2007 and 2008. Subtraction can be sneaky that way.

Now this is a biological problem (the migratory patterns of a human being, me), and I used a calculator on my computer to solve it. That makes it an example of bioinformatics. Everyone says bioinformatics is really hard, but I didn’t find it that bad at all. Of course it depends on the methods you use. For example, bioinformaticians sometimes use Markov models or Monte Carlo simulations, but those would have been a whole lot harder, especially since I don’t know what they are. So I just used basic subtraction. (By the way, I have to do the same thing when somebody asks me how old I am.)

Russ, puzzling over a problem in bioinformatics

Of course these results don’t have any statistical validity, so I added this:

(2014 –  2007) + 1 = 8    (p < 0.05)

I know even less about statistics than bioinformatics, but I do know that statisticans always put a p in there somewhere, and it’s always greater or lesser than some other number, so there you go. If you want to know more about this, we’ll be hearing later from a biostatistician, Jo Røslien, and I’m sure he’ll be glad to explain it to you.

Eight trips to Oslo, wow. There aren’t many places in the world I’ve been eight times. I’ve probably been home a few more times than that. If you ask my wife, she might not agree, but then, she’s not a bioinformatician.

When you’ve been to a place eight times you’re practically a native. That’s how I feel about Oslo. Of course, I don’t speak a single word of Norwegian, which makes me a strange sort of native. Sort of a foreign native. Still, Oslo has some surprises even for a native. For example, when I arrived yesterday, Oslo looked DIFFERENT. Was there more than one Oslo? Had I flown to the wrong one? It took me a while to figure out the problem, but I finally did: all my other trips have been in DECEMBER. I don’t know if you’ve noticed this, but it’s DARK in December. I arrive in Norway, get off the plane, and I just automatically say, “Would somebody please turn on the lights?”

In science, the hardest part of finding an answer is sometimes clearly identifying the problem, and that was certainly true in this case. Once I knew that Oslo was too bright, there was a simple solution – just put on some sunglasses.

So once again, thank you for inviting me to Oslo, where I feel like a native, but a special kind of native, who only comes out in the dark and has poor communication skills. Maybe a native Norwegian bioinformatician. Or a native Norwegian vampire. Or a native Norwegian bioinformatician vampire. If you have any of those in Norway, that’s exactly how I feel.

* * * *

Science communication is serious business, and I do have some serious things to say about it, and I promise I will. But one of the most important things I have to say about it is that we ought to have more FUN at it. I’ve been a professional science communicator for 17 years. It’s a great job, but sometimes it’s hard. You run up against a lot of walls. If you could see them coming, you could avoid them, but they’re sneaky, and they’re mostly invisible. You’re writing an article, or you’re talking to someone, and boom, you run against an invisible wall, and you go flying on your ass. Over and over again.

I’m going to talk quite a bit about invisible walls today. These walls are in the minds of scientists, and in the minds of science communicators, and in the minds of the people we’re trying to talk to. I’m going to talk about ways to make invisible walls visible, and what that means for science and science communication.

But first let me say that if you see somebody run along, and he bangs into an invisible wall and falls down, and then he gets up and starts running and it happens all over again, you’re probably going to laugh. It’s not so funny when it happens to yourself… Well, why not? Why not laugh at ourselves a little bit? So I decided to start a CRUSADE to make science communication more FUN.

Now the crusades of history involved huge armies of religious people setting out to take over a foreign country. I’m not religious, and my crusade is very small; in fact, there are no followers except for a few people who read my blog and may be seriously disturbed. But a lot of great ideas start small. That’s also true of bad ideas, I know. The trick is to tell the difference. How? Only time will tell. I’m still at the very beginning of my crusade, the early planning phase, sort of the grant application phase. For example, I haven’t picked which country this crusade will invade. Maybe Norway? We’ll see.

So today I’m going to talk about invisible walls, and making science communication fun, and hopefully say something useful along the way. We’ll see.

* * * *

Sometimes I think science communication is difficult because we’ve gotten ourselves into a box and we need to get out. This inspired the title of my talk, LETTING SCIENCE COMMUNICATION OUT OF THE BOX, in case you’ve forgotten. There’s also a CAT in the title, which you may also have forgotten, but that’s okay, I’ve just reminded you.

I’m sure most of you realize I’m alluding to a famous experiment proposed by the physicist Erwin Schrödinger. In fact, one of the speakers this afternoon, Chris Vøløy, is involved in a project called Schrödinger’s cat. Actually his project is called “Schrødingers katt” , which is Norwegian, so I might be translating it wrong. For all I know, in Norwegian this might mean “Einstein’s parrot.” Chris, are you here? Did I get this right?

Schrödinger’s cat is a THOUGHT experiment, which means the cat is an imaginary cat. Now if a biologist were to do an experiment with an imaginary animal, he’d face a challenge getting his paper published, but in physics you can get away with these things.

Nobody has ever done Schrödinger’s experiment, or if they have, they won’t tell you about it, because it would violate all kinds of regulations about the ethical treatment of animals. Okay, it’s a metaphorical cat, but even metaphorical animals deserve some respect.

Erwin Schrödinger and his cat

Schrödinger’s experiment also violates all kinds of rules of common sense (for example, the difficulty of getting a cat into a box). Again, in a thought experiment that doesn’t matter. For example, in a thought experiment I could be the King of Norway. Or the Queen of Norway.

Anyway, Schrödinger’s experiment with the cat is interesting for science communication because it’s unethical, illegal, probably impossible, and on top of that, NOBODY UNDERSTANDS WHAT IT MEANS. Well, it meant that Erwin Schrödinger didn’t like cats very much. But other than that nobody really understands it. And yet 79 years later here we are still talking about it. If that’s not successful science communication, I don’t know what is.

Basically the experiment involves locking up a cat in a steel box with one atom of a radioactive substance that would decay after a certain period. Now it’s a bit irritating that Schrödinger leaves some details of the experiment vague, and others he explains in great detail. For example, he doesn’t mention what type of atom you should pick, or how you capture only one atom, or how you know you’ve got the right one. Another detail he doesn’t explain is whether in this experiment, he is the King of Norway or not.

But he does go into detail about the placement of the atom in a Geiger counter. Once everything is in place, if the atom decays, “the counter tube discharges and through a relay releases a hammer that shatters a small flask of hydrocyanic acid.” In case you don’t know, that’s a particularly nasty type of poison. Anyway, you’re not supposed to look into the box. The question is this: At some point, the half-life of the radioactive substance, there is a 50 percent chance that the cat will be alive. Schrödinger doesn’t tell us what the probability is that the cat will be dead, but there’s a formula for this somewhere; you can find it on the Internet.

With all of this Schrödinger wanted to make a specific point. Schrödinger says that until somebody opens the box, the cat is in an indeterminate state. It’s not really alive and not really dead. Actually he puts it a different way, and if you ask me, it’s a pretty disgusting. He says, “The living and dead cat (pardon the expression) [will be] mixed or smeared out in equal parts.”

This is why I said it’s important to choose the right radioactive substance. If you have a substance that decays in one hour, that’s fine. But suppose you pick a substance that takes longer to decay, a lot longer. Maybe weeks or years. I don’t know if you’ve ever kept a cat in a box for a really long time, but I’m telling you, it smells bad. It smells bad whether the cat is dead or alive, but those are different kinds of bad smells. You’ll definitely know. And even if you don’t, the cat will know.

Now as I said, this is a thought experiment, so we don’t have to care that much about the methods, or the results, or the discussion. Today Schrödinger would never get this published, except on a blog, or in The Journal of Impossible, Ridiculous, and Pretty Disgusting Thought Experiments. Well, maybe not. I know the editors of that journal personally and they might not even publish it.

In fact, there is a group of people in California who are actually doing this experiment, or at least an experiment that’s pretty close. I’m sure you’ve heard of them. I’m talking about those companies that offer cryopreservation. You pay the company a lot of money and when you die, they will wrap you up in tin foil and freeze you. The idea is that in 100 or 200 years, or 1000 years, or a million years, scientists will be able to cure the disease that killed you. They’ll even be able to cure it after you’re dead, which is pretty wonderful.

This is supposing, of course, that in one million years the company will still exist, and that California will still exist, rather than sinking in an earthquake, and that in one million years there has never been a power blackout in California. Right. In a state that elected Arnold Schwarzenneger governor. Good luck. In fact, there have already been blackouts. You have to hope the company has some big batteries somewhere, with a lifetime somewhat longer than the battery on your laptop.

Anyway, if the electricity has stayed on all the time, and if somebody remembers the human popsicles stored in the freezer in the basement, and if science learns to cure cancer and Alzheimer’s disease and develops some sort of nanotechnology to repair the damage that being frozen for a million years causes to a human body, maybe those people will be revived. You can pop them into the microwave. But first remove the aluminum foil. You shouldn’t put aluminum foil into a microwave. I’ve tried, and the results are not pretty.

The frozen people will probably wake up in some sort of Star Trek universe, where there’s all kinds of new technology they have to learn. The iPhone one million point five. People who use Macs will have an advantage. I’m sure we’ll still have Macs in a million years. But we won’t have PCs. PCs will have become extinct, like dinosaurs, maybe because of an asteroid strike.

Well, until all that happens, those people in California are in a state like that of Schrödinger’s cat. Except for the part about being mixed or smeared out. That’s might happen if the electricity goes out. But if everything goes well for a million years, we hope they’ll be reasonably intact. It’s hard to say. They’ll probably be fatter – water swells a bit when it turns to ice – but they’ll have been transformed from a state of death to a state of life. So yes, I guess you can say that currently, those people are in an indeterminate state like Schrödinger’s imaginary cat.

The Jentsch lab and the FMP Screening Unit identify a long-sought channel that helps cells reduce their volume

Some important scientific questions resist a solution for years, until a new technology appears that seems to put an answer within reach. Success depends on creativity – finding a way to translate an old question into a form that can be handled by the new method – but also on timing. And a bit of luck never hurts.
About four years ago Thomas Jentsch thought that the time had come for an all-out assault on a question that had teased biologists for decades. The campus had been acquiring instruments that might be the key to the solution. But the clock was ticking; other labs had the technology, too, and were interested in the same question: How do cells reduce their volume?

All types of cells undergo alternating phases of expansion and shrinkage as they divide, specialize, migrate, and cope with changes in their surroundings. In a liquid environment without any barriers, particles move from areas of high concentration to low. This applies also to water “particles”, which tend to diffuse until they have reached the same concentration everywhere. Water will flow from regions of high concentration (where it is diluted by a low concentration of osmolytes like ions, the constituents of salt, or other particles) to areas of low water concentration (where it is diluted by a high concentration of salt or other particles). So if cells are exposed to a hypoosmolar solution (which contains fewer osmolytes), water will flow into the cell, which now contains more osmolytes than its extracellular environment. But because the cell is enclosed by a membrane, the particles within it cannot simply be diluted to reach an equilibrium with extracellular concentrations. Instead, osmotic pressure will build up inside.

The same accumulation of internal water can occur when the number of internal particles increases through a breakdown of organic molecules, like glycogen to sugar, and cells may expand until the membrane is stretched to its limits. In order to keep from bursting, the cell needs to lose particles such as chloride or potassium ions, or organic osmolytes such as the amino acid taurine.

The loss of these particles decreases their internal concentration, which eventually becomes lower than in the external medium. This leads to a reversal of water flow, cell shrinkage and a recovery of the previous, normal cell volume and shape – called regulatory volume decrease. This volume decrease was known to be associated by a loss of negatively charged chloride ions (anions) from the cell through a channel.

Many aspects of the transport of chloride and other ions are well understood thanks to years of research on the part of Thomas’ lab and many others. This type of work has exposed hundreds of types of ion channels in cell membranes. These pore-like structures are composed of proteins woven through the membrane in a way that allows them to open and close. Channels are highly specialized to admit specific ions or other particles in response to changes in electrical charge, pressure, or other conditions. Their behavior is usually regulated by intricate biochemical networks. Most ion channels are so essential that any flaw in their components or their regulation can cause serious diseases – Thomas’ lab has established many such connections.

Much less was known about the outward passage of anions like chloride and other particles as cells shrink. Scientists postulated the existence of a specialized channel and had even given it a name: the volume-regulated anion channel, or VRAC. “VRAC was a hot topic,” Thomas says. “Hundreds of papers were being written about it and it was the subject of major international conferences.”

Despite all the interest, and the fact that the biophysical properties of the channel and some of its important functions were known, no one was able to actually find the channel protein. Occasionally a lab claimed to have identified a protein that belonged to it, but one by one, these candidates were discredited. While a number of drugs interfered with the channel’s behavior, they also disrupted other cellular processes. Without identifying a specific protein, scientists could understand neither these effects, nor the way swelling triggered changes in the channel’s behavior.

Part of the interest – aside from the fact that the channel served an important basic function in all types of cells – arose from observations that it, too, seemed to be linked to serious diseases. In any case, Thomas doubted that VRAC could hide much longer. The sequencing of the genomes of humans and other organisms had led to the development of technologies that permitted scientists to scan the DNA sequence for molecules with specific functions. Recent papers by other groups hinted that they were using these methods to search for the channel.

“Identifying the VRAC would finally give us a handle on some of these questions,” Thomas says. “Basically, it would open up an entirely new field.”

* * * * *

Until fairly recently, the study of gene functions almost always required an arduous, molecule-by-molecule approach. The earliest methods involved forward genetics: a scientist found an organism with a specific variant of a feature or a defect, then studied patterns of inheritance to establish that a single gene was involved. You might not identify it for years, or decades, but at least you knew it existed.

Modern biochemistry made it possible to do reverse genetics, through which scientists interfered with a specific gene and then studied the consequences for an organism. Most of these approaches depended on physically removing or altering genes, and thus eliminating the proteins they encoded.

The 1990s brought a powerful new alternative based on introducing artificial molecules called small interfering RNAs (siRNAs) into cells. siRNAs are composed of nucleotides, like other RNAs and DNA. This means that strands with complementary sequences will dock onto each other, which is the reason for the double-stranded helix structure of DNA. RNAs are usually single-stranded, but if an siRNA docks onto a complementary mRNA it produces a short, double-stranded region. Cells normally take notice and dismantle the molecule before it is used to make proteins. So artificially introducing siRNAs into cells has become a new, effective way of “knocking down” particular molecules by interrupting the flow of information from gene to RNA to protein.

Since the first applications of siRNAs in plants and simple model organisms, researchers have adapted them for use in human cells. The completion of the human genome revealed the entire set of mRNAs encoded in it, and scientists working in companies began constructing vast “libraries” of siRNAs to target them all. Theoretically this could be used to block the production of every human protein, one-by-one. Along the way, you would probably hit a molecule required for a particular function – such as the VRAC – and shut it down.

Humans have over 22,000 genes, meaning that the project would require at least that many experiments – actually the number would be much higher. There is no way to predict with certainty that a particular siRNA will work reasonably well within a cell. The way to get around this was to develop multiple siRNAs for each target and then challenge cells with two or three different versions; one of them would usually work.

But 22,000 experiments – let alone two or three times that number – could not be performed by hand. Robots and automation would be required at each step: to prepare samples, perform experiments and collect the data, and sophisticated software to analyze the results.

In the genome age, the technology and software needed for genome-wide siRNA screens began coming together, leading to several successful projects that have produced fascinating new insights into the functions of genes.

Several years ago the FMP created a Screening Unit, now jointly operated with the MDC, and acquired high-throughput, automated equipment to manage such large-scale projects. Headed by Jens von Kries, the facility has made significant contributions to a number of campus projects, many drawing on the tens of thousands of compounds that the unit has collected from researchers, pharmaceutical companies, and other sources. A typical use of this vast library is to challenge cells with substance after substance, hoping to disrupt a specific process in cells – with the aim of further developing the “hit” into a research tool, possibly even a drug. Chemists associated with the Unit then step in to tinker with a successful substance to strengthen its effects, make it less toxic for cells, or lend it other desirable properties.

The Screening Unit had also acquired a library of siRNAs that targets every known gene and then, through grants including a successful application from Thomas to the European Research Council (ERC), also a machine called FLIPR that was needed for following the time course of hundreds of experiments in parallel. So the necessary tools were on hand to search for VRAC components. “But first an extremely reliable experimental procedure had to be developed to detect the opening of the VRAC channel upon cell swelling,” Thomas says. “Any test that will be carried out tens or hundreds of thousands of times has to deliver clear, dependable results.”

Postdoc Tobias Stauber and PhD student Felizia Voss took the first steps toward developing a robust experimental protocol. This required almost two years, and as Thomas says, “There was no guarantee that we would find VRAC, even with this whole-genome approach, and certainly no guarantee that we would find it first.” Failure would have been particularly hard on Felizia, who was devoting her entire time to the project, and other PhD students who had become involved. Positive results would provide an impressive story for their dissertations; no one wanted to consider the alternative.

Tobias and Felizia began by growing cultures of human embryonic kidney cells – a favorite model for genetic research because the cells readily take up foreign molecules such as siRNAs. First they equipped cells with a yellow fluorescent “reporter” protein that would reveal whether cells changed their concentration of anions upon swelling. The cells were placed in a diluted medium in the presence of the ion iodide, which entered the cells through the opened VRAC channel and quenched the fluorescence. Although VRAC opening leads to a net loss of cellular chloride and volume regulation, the addition of external iodide, which is not present in the interior of the cell, leads to a net influx of iodide – channels are like “holes” that allow movement in both directions. The basic idea was that an siRNA that blocked the production of VRAC within the cell would abolish, or at least reduce, the loss of fluorescence upon exposure to a diluted medium containing iodide.

“This entire approach depended on an assumption that simply might not turn out to be correct,” Thomas says. “If a single specific molecule were required for VRAC, we might find it in the experiments. But if several molecules formed VRAC together, and each of them could be functionally replaced by another one, we might never find it with this approach. In fact, we discovered that VRAC has five components, and only one is absolutely required – we were lucky that only four of the subunits can replace each other.”

The scientists optimized the transfection of siRNAs and other aspects of the experiments, such as the number of cells, and established appropriate controls. Once this had been accomplished, it was time to scale up the experiment for a genome-wide search for VRAC components, trying to eliminate one essential component of the channel.

Now Katina Lazarow, a postdoc with the Screening Unit, stepped in to adapt the procedure to the instrumentation. Each step of the pipeline of experimentation and analysis had to be adapted to the specific question the scientists were trying to answer. This required many months of intense work – often through the weekends. The actual screen would also take about two months of work, once again including weekends.

* * * * *

Such a large experiment required automation for the preparation of cell cultures, introducing a different siRNA into each one, then capturing images that would show whether the intervention had any effect on VRAC. These steps were performed in a specialized piece of machinery called the FLIPR, acquired second-hand with funds from Thomas’ grant and the Screening Unit.

The machine has become an important addition to the Screening Unit because it allows scientists to prepare experiments in all 384 “wells” of a assay plate in parallel, and follow changes in fluorescence over time, also in all the wells in parallel. The scientists needed to follow the exact time course of a decrease in fluorescence, because it is a measure of the flow of anions through VRAC.

Applied to the whole genome, this produced more than 130,000 fluorescence curves to analyze – a step that obviously required bioinformatic analysis. Miguel Andrade’s group at the MDC took on the task.

“We specified the parameters necessary to distinguish possible VRAC components from all the other proteins that didn’t affect the transport of chloride ions out of the cell,” Thomas says. “In the end we were left with a list of about 200 proteins that might be involved.”

More analysis and experiments were required to whittle the list down to just a few – hopefully just one – candidate. Part of this could be done by a computational analysis of the proteins’ sequences. The main goal was to find molecules with patterns indicating they were likely inserted into membranes. But other types of molecules might be worth investigating as well. A biochemical signal – passed along by a specific protein – might be required to operate the channel, even if that protein wasn’t a direct component of VRAC. Blocking it might shut down the channel just as well as eliminating one of the membrane proteins. The data collected in the experiments may eventually shed light on these processes. “We now have a treasure trove of data that remain to be analyzed; first we were concentrating on candidates for channel components,” Thomas says.

Felizia, Tobias and Thomas took on the list of candidate membrane proteins and began sorting through everything that was known about the functions of the candidates, from other experiments. The aim was to distinguish promising proteins from those that were less so – “And you had to be careful not to discard any molecule that might turn out to be the real one!” Thomas says.

After this analytical effort, over 80 proteins remained for one-by-one investigation through more experiments. Using the same experimental protocol, they challenged the cells with new siRNAs directed against those same 80 genes. The scientists dug in for another period of hard work.

This time, disruptions of only one molecule – a protein called LRRC8A – continued to interfere with VRAC. “So far we had only covered one aspect of VRAC function, the cells’ uptake of iodide,” Thomas says. “Now we wanted to explore another aspect – to measure its effects on electrical currents. We did this using a method called patch-clamp.” Two PhD students from Thomas’ lab, Florian Ulrich and Jonas Münch, joined the team and worked for about a year on the analysis of VRAC. They discovered that the knock-down of LRRC8A also reduced chloride currents. This meant that LRRC8A was either a direct component of the channel, or it exercised a firm control on VRAC functions. Thomas’ lab began the next round of experiments to find out.

* * * * *

“Proving that LRRC8A protein was a direct component of the channel required a number of steps,” Thomas says. “Upon a closer look we saw that it fulfilled a number of important criteria. First, it was expressed by many types of cells in the body, all of which need to regulate their volume and are thought to express VRAC. And a study of its sequence showed that it had regions that would be embedded in membranes – but was it really present in the plasma membrane that encloses cells?” The scientists generated antibodies that attached themselves to LRRC8A and observed under the microscope that LRRC8A indeed moved to its expected position at the outer membrane.

So far, so good – but was LRRC8A the channel itself? The scientists overproduced the protein in cells and measured currents activated by swelling. “Of course we had hoped that this would boost swelling-activated currents way beyond those in normal cells,” Florian says. “But were dismayed to see that rather than increasing currents, overproducing LRRC8A actually decreased currents!”

Thomas had an explanation for this behavior: “Channels are often composed of multiple proteins woven together in the membrane,” he says. “LRRC8A might form a channel together in a complex with other proteins, and if we had too much of LRRC8A, it may assemble with these other proteins at the wrong ratio, leading to complexes that cannot pass currents.”

What might those proteins be? Obvious candidates were molecules that were very closely related to LRRC8A. The cells of humans and other animals have many such “homologs”: far back in evolutionary history, in ancestral organisms, biochemical mistakes occasionally produced multiple copies of many genes. In the species in which they originally occurred, the copies would be redundant. That meant they could be lost again, which often happened as the genes naturally underwent mutations.
In some cases, however, spontaneous mutations enabled the copies to develop new functions. So the human genome contains four other versions of LRRC8 (labeled LRRC8B through E) that may function in a similar fashion.

“Databases containing information from other experiments showed that these molecules, too, are widely expressed in various types of cells,” Thomas says. “When we began to study these molecules individually, by introducing them into cultured cells, we noticed something interesting. Most of them remain inside the cell, in the cytoplasm. But if we introduced them along with LRRC8A, they move to the membrane.”

This indicated that LRRC8A binds to these related LRRC8 proteins, and that certain combinations of LRRC8s might be required to create the channel. Another round of experiments removed and restored various combinations of the proteins. Since siRNAs usually achieve only a partial blockage of a given protein, and might affect other molecules than the intended target, it was necessary to turn to another method. Here the scientists used a new technique called CRISPR-Cas, which interferes with genes directly at the level of DNA rather than its messenger RNA products. CRISPR-Cas allows scientists to make a physical cut in the genome and eliminate a specific gene. The results replicated the findings of the siRNA experiments and importantly showed other LRRC8s were involved in VRAC, but always in combination with LRRC8A.

The scientists found that each of the other LRRC8 proteins, B-E, could be deleted alone without abolishing VRAC currents. But when B-E were eliminated together, VRAC currents were gone just as if LRRC8A itself had been removed. “This showed that LRRC8A needs at least one of the other subunits to form a channel, but that LRRC8B through E are redundant – in other words, they can replace each other,” Felizia says. “And we even generated a cell line lacking all the forms A through E. Currents could only be restored when expressing A with at least one of the other family members.”

Importantly, different combinations of proteins yielded channels with different biophysical properties. “These are intrinsic channel properties,” Tobias says, “which demonstrates that LRRC8 proteins are not just involved in the signaling system that opens the channel upon cell swelling, but are an integral part of the channel itself.” This observation solved another enigma in the field – VRAC currents seemed to vary in different cells and tissues. “Our work now explains the variability of currents because different cells in the body make different amounts of these proteins. The different combinations may serve cell-specific functions.”

The project also provided hints toward answering another interesting question about channels involved in cell shrinkage. “The loss of volume involves expelling small organic molecules such as taurine,” Thomas says. “There was a controversy among experts – some claimed that these molecules exited via the VRAC, whereas others thought they might use yet another channel.”

Further experiments revealed that VRAC accomplishes both functions. The lab used to a radioactive form of taurine whose export from the cell could be tracked. Darius Lutter of Thomas’ group adapted a test that would measure its efflux, and the scientists found that taurine’s behavior aligned with the presence or absence of various LRRC8 molecules and VRAC behavior.

“All in all, these experiments reveal that the channel is directly constructed from combinations of LRRC8A and other LRRC8 forms,” Thomas says. “They give us the first clear route to explore the mechanisms by which cells shrink and shed particles in response to swelling. Those functions are crucial in the context of all types of cells, and this work provides the basis for understanding how they become defective in a number of serious diseases.”

Even with the well-coordinated collaboration and intensive efforts on the part of Thomas’ labs and the Screening Unit, the group’s search for the VRAC took four years. But the race would only officially be won if his team was first to publish on the subject. The scientists quickly wrote up a paper on the project and submitted it to a major journal.

All the way along, Thomas had been worried that other groups might be working on the problem; after four years, getting scooped would be a hard blow. Those concerns turned out to be justified. On the very same day that the article appeared in Science, another lab published a paper in the journal Cell that identified LRRC8A as a component of VRAC. That work did not, however, demonstrate that the “A” form had to be combined with another LRRC8 to create the channel.

“Our work puts the whole field of cell volume regulation on firm ground,” Thomas says. “The signals that connect an increase of cell volume to the opening of the VRAC are enigmatic, and we now have a handle to investigate them. We have suspected that defects in VRAC are involved in a number of diseases, and now we can develop mouse models to confirm those roles and look for other functions of the channel.”

Reference:
Voss FK, Ullrich F, Münch J, Lazarow K, Lutter D, Mah N, Andrade-Navarro MA, von Kries JP, Stauber T, Jentsch. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. TJ.Science. 2014 May 9;344(6184):634-8. doi: 10.1126/science.1252826. Epub 2014 Apr 10.

The Berlin magazine Occulto will soon publish my short story “Remote sensing” – about a young man and his grandfather, a dowser. This is the first time they’ve published a short story. To order a copy of this very interesting magazine, visit:

http://www.occultomagazine.com/shop.html