Jan. 27, 2003 Right now, in your body, tiny pumps in the fatty membranes surrounding all your cells are hard at work pushing select charged ions, such as sodium, potassium or calcium, through those membranes. Like a water pump in a high-rise apartment building overcoming the force of gravity to move water up to a tank on its roof, these ion pumps work against "electrochemical gradients" to transport ions from one side of the membrane to the other.
Other tiny machines, called ion channels, also embedded within membranes, are like the apartment building's faucets: they harness the energy stored in this "uphill" process by allowing ions to rush back "downhill" across the cell membrane through the channels' open pores.
Such movements of ions into and out of a cell form the basis for the transmission of signals in the brain and heart as well as other tissues of the body that allow you to breathe, move, think, digest food and pretty much function in all respects.
But, while decades of research have illuminated a great deal about the structure and function of ion channels, ion pumps have remained stubbornly resistant to scientific inquiries.
Now, in the Jan. 21 issue of Proceedings of the National Academy of Sciences, researchers at The Rockefeller University report using palytoxin, a deadly coral-derived toxin, to pry open perhaps the ion pump's deepest secret: that it is essentially a more elaborate version of an ion channel.
"The 'pump as channel' model is actually a very simple way to look at the function of ion pumps," says lead author Pablo Artigas, Ph.D., a postdoctoral associate in David C. Gadsby's laboratory, adding that this latest research has revealed that "nature has once again figured out the simplest solution."
"Since the late 1950s, a handful of scientists have imagined pumps and channels as sharing some similarities, but this is the first time we've been able to establish this experimentally," says senior author Gadsby, Ph.D., head of the Laboratory of Cardiac and Membrane Physiology at Rockefeller. "By interfering with the pumps' normal conformational changes, the coral toxin essentially turns them into channels."
The researchers specifically studied the sodium/potassium pump, the most common (and arguably most important) of the human ion pumps. Its impaired activity is believed to underlie high blood pressure and it is the target of digoxin, one of the most widely prescribed drugs for heart disease. A closely related ion pump, the hydrogen/potassium pump, controls the production of stomach acid and is targeted by new antacid drugs, such as Prilosec.
A better understanding of the molecular workings of ion pumps may ultimately pave the way for better treatments for hypertension and heart failure, and possibly other disorders.
"We hope that palytoxin will give us a more detailed picture of the molecular mechanisms underlying the function of one of our body's most essential and remarkable microscopic machines - the sodium/potassium pump," says Gadsby.
Gated community of the cell
Unlike ion channels, which permit the flow of just one specific type of ion, most ion pumps transport different kinds of ions in opposite directions across cell membranes. The sodium/potassium pump, for example, passes three sodium ions out of the cell for every two potassium ions pumped into the cell.
Just how this "uphill" ion exchange is accomplished without "downhill" leakage was first proposed in the late 1950s in the "alternating-access" model, which hypothesized that ion pumps might allow the entrance and exit of ions at only one side of the membrane at a time, rather like a revolving door. In this model, a pump acts like an ion channel with two gates, one at either end, that are constrained to open alternately but never at the same time - one gate must close before the other can open. But no concrete evidence in favor of this four-decade-old model existed - until now.
Presto, you're an ion channel!
To trick the sodium/potassium pump into revealing its true "ion channel" nature, Artigas and Gadsby applied palytoxin to cells and used electrophysiology - specifically the "patch-clamp" technique - to detect its effects. The patch-clamp technique has enabled scientists to detect electrical currents produced by the flow of ions through a single channel - yet it hasn't been able to detect a single working pump molecule.
This is because while a channel easily passes ions across the cell membrane at a rate of up to hundreds of millions of ions per second, making a current that is readily detectable, a pump has to work hard to move only hundreds of ions per second - too few to produce a measurable signal. So, when the researchers added palytoxin to a single pump and observed its electrical signal immediately go from zero to almost a trillionth of an ampere, they knew that what had been a hardworking pump had indeed been transformed into a free-flowing channel.
"What the toxin does is to allow the pump's two gates to be open at the same time," says Artigas. "So the key to the pump's normal function is the strict coupling between its gates. The gate at one end must know whether the gate at the other end is open or closed, which means that the two must communicate."
In addition, because the researchers can now, with the help of palytoxin, zoom in on a single sodium/potassium pump for the first time, they can ask all sorts of new questions. For example, scientists do not know the location of the pump's gates within its protein structure. The new approach should allow them to solve this mystery as well as answer other detailed questions about how the pump works.
Toxins deadly useful in lab
The new study is not the first time that researchers have turned to natural toxins to better understand the complexities of ion pumps. Beginning in the 1950s, scientists have taken advantage of the inhibitory properties of both digoxin, the widely prescribed heart drug isolated from the foxglove plant digitalis, as well as a close relative called ouabain, a Native American arrowhead poison. Like straitjackets, ouabain and digoxin hold the sodium/potassium pump in a fixed conformation. In 1989, Gadsby and colleagues used ouabain to prove the sodium/potassium pump's fixed stochiometric ratio of three sodium ions to two potassium ions. And, more recently, they used ouabain to show that the three sodium ions are expelled one at a time by three small, sequential changes in the pump's conformation.
Artigas and Gadsby's latest research puts palytoxin on this important, historical list of nature's unintended tools.
Founded by John D. Rockefeller in 1901, The Rockefeller University was this nation's first biomedical research university. Today it is internationally renowned for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. A total of 21 scientists associated with the university have received the Nobel Prize in medicine and physiology or chemistry, 16 Rockefeller scientists have received Lasker Awards, have been named MacArthur Fellows and 11 have garnered the National Medical of Science. More than a third of the current faculty are elected members of the National Academy of Sciences.
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