26 October 2008

Palytoxin and zoanthids of death

These pretty (and very common) colonial anemones or zoanthids (Order Zoanthidea) can harbour one of the most potent natural toxins.
Various kinds of colonial anemones (Order Zoanthidea)
When first discovered, the toxin was so potent that it killed mice within seconds. There is a case of Palytoxin poisoining after contact with a zoanthid in an aquarium through skin injuries on the finger. This is why we should NOT touch our marine life, and if we do, certainly never to put the finger in the mouth or eye after that.

There is even a case of a professor who tried to poison himself with palytoxin after finding out that his wife had an affair. Sigh.

Palytoxin disrupts the transfer of ions in our cell membranes, a process that is responsible for transmission of signals in the brain and heart as well as other tissues of the body that allow us to breathe, move, think, digest food and pretty much function in all respects.

Using palytoxin helps scientists better understand how our ion pumps work and may ultimately pave the way for better treatments for hypertension and heart failure.

Scientists had in the past also used natural toxins to better understand and treat heart disease. These include digoxin, isolated from the foxglove plant digitalis, as well as a close relative called ouabain, a Native American arrowhead poison.

Below are more stories of the discovery of palytoxin and the eight-year effort to synthesise the humungous molecule.

Deadly Coral Toxin Exposes Ion Pump's Deepest Secret
ScienceDaily 27 Jan 03
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.

Science: The making of a molecule in a billion billion
John Emsley, New Scientist 27 Jan 90
A TEAM of 22 chemists at Harvard University has taken eight years to make an exact copy of a huge natural molecule, a poison produced by a marine coral. The poison, called palytoxin, has the formula C129H223N3O54 and a relative molecular mass of 2680. For a chemist, synthesising it is equivalent to building the Great Pyramid.

Palytoxin is made by the coral Palythoa toxica. It is one of the most poisonous chemicals known, almost as deadly as some of the toxic proteins produced by bacteria, insects and plants.

Chemists first deduced the structure of palytoxin in 1981, revealing a mammoth molecule. Identifying the exact pattern of rings, chains and groups was a triumph of the chemist's art.

The molecule has 64 carbon atoms that can give rise to alternative structures; it can also arrange itself in various ways about six of its double bonds. In fact, there are over a billion billion possible forms of palytoxin. Nature makes only one.

The story of palytoxin began in 1961 when a biologist, Albert Banner, of the Hawaii Institute of Marine Biology, came across the name Limumakeohana in a Hawaiian-English dictionary. The name means 'deadly seaweed of Hana'.

Hana is a town on the island of Maui. The seaweed reputedly grew in a tidal pool near the town, where the locals once used it to poison the tips of their spears. Local people in Hawaii believed that the seaweed was cursed and refused to reveal its location.

The scientists eventually tracked the pool down: it turned out to be no larger than an ordinary bathtub. The organism growing there was not a seaweed but a soft coral. It was certainly deadly; treating the coral with alcohol extracted a substance so potent that it killed mice within seconds.

There followed years of work to discover the chemical composition of the active component, which scientists named palytoxin. Two research groups published its structure in 1981. The surprise was that the molecule was so big.

Undaunted by this finding, Yoshito Kishi and his colleagues at Harvard University set out to make palytoxin in the laboratory. Last year, they completed the mammoth task and announced their triumph in two papers in the Journal of the American Chemical Society (vol III, pp 7525 and 7530). Kishi solved the problem by identifying eight subunits that chemists could make separately and join together to make the complete molecule. Making the subunits took three years; joining them together took a further five.

The problem that Kishi faced was how to protect the reactive centres on these units while assembling them into the final molecule. Organic chemists have an armoury of protective groups that they can attach to parts of a molecule, which render them insensitive to chemical attack during the main reaction. It is easy to remove the protective groups later.

The skill is in choosing which protective groups to use. Kishi wanted to protect over 40 centres, most of them hydroxyl (OH) groups, and he used eight groups.

Having made the subunits, protected them, joined them together and removed the protective groups, the Harvard team still had to show that the molecule they had made was identical with that of natural palytoxin. They did this by measuring the distance between the two ends of the molecule. Any difference between the natural and the synthetic molecule at one of the 64 nonsymmetric carbon atoms or six double bonds would have produced a different distance.

To check this measurement, Kishi and his colleagues attached groups, capable of transferring energy when stimulated with ultraviolet light, to each end of the synthesised molecule and to each end of the natural one. The transfer of energy is very sensitive to the distance between the two groups. The distances matched.

Palytoxin: A New Marine Toxin from a Coelenterate
Richard E. Moore and Paul J. Scheuer
Department of Chemistry, University of Hawaii, Honolulu 96822
Science 30 April 1971:
Vol. 172. no. 3982, pp. 495 - 498
from Science of the AAAS
Palytoxin has been isolated from the zoanthids "limu-make-o-Hana" (Tentatively identified as Palythoa sp.) as a noncrystalline, chromatographically pure entity. Apart from polypeptide and protein toxins, it is the most highly toxic substance known, with a lethal dose (LD59) in mice of 0.15 microgram per kilogram by intravenous injection. Unlike the potent toxins batrachotoxin, saxitoxin, and tetrodotoxin which have molecular weights of 500 or less, palytoxin has an estimated molecular weight of 3300 and contains no repetitive amino acid or sugar units.

A case of palytoxin poisoning due to contact with zoanthid corals through a skin injury
Katrin Hoffmanna, Maren Hermanns-Clausenb, Claus Buhla, Markus W. B├╝chlera, Peter Schemmera, Dietrich Mebsc and Silke Kaufersteinc
Toxicon, Volume 51, Issue 8, 15 June 2008, Pages 1535-1537
on the ScienceDirect website
A case of human poisoning by palytoxin after contact with zoanthid corals (Parazoanthus sp.) in an aquarium through skin injuries on fingers is reported. The clinical symptoms include swelling, paraesthesia and numbness around the site of the injury spreading over the arm, but also signs of systemic poisoning such as dizziness, general weakness and myalgia, irregularities in the ECG and indications of rhabdomyolysis. Symptomatic treatment consisted of infusion of physiological fluids. The patient recovered within 3 days. Analysis of the zoanthid coral involved revealed extremely high concentrations of palytoxin (between 2 and 3 mg/g).

More links
  • Professor plotted suicide by poison
    Linus Gregoriadis The Telegraph 19 Jun 01
    A professor tried to obtain palytoxin to kill himself after learning that his wife was having an affair.

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