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Giving toxins a long lead
Ion channels and cell surface receptors yield to precise form of in vivo manipulation
BY LYNN LOVE
Inspired by toxins found in poisonous snails, snakes and spiders, Rockefeller researchers have created a new system that can alter the physiology of cells, fundamentally changing their behavior.
Rockefeller research associate Ines Ibanez-Tallon, who as of September heads her own lab in Berlin’s Max-Delbrück-Center for Molecular Medicine, and her colleagues in Professor Nat Heintz’ laboratory of Molecular Biology devised a way to “knit” together a lipid anchor, a variable length peptide tether, and a naturally occurring peptide toxin so that they can selectively block a wide variety of neurotransmitter receptors and ion channels.The system, called “tethered toxins,”may even work to block hormones and other secreted proteins in the nervous system and elsewhere.
“The applications of this system are so vast; the challenge for us is to determine which experiments to do first using tethered toxins,” says Heintz.
The path to the new system began in 1999, when Julie Miwa, a postdoctoral associate in Heintz’ lab, realized that one of the brain’s native molecules (which she and her colleagues named lynx1) closely resembles the large numbers of lethal toxins found in animals. Her analysis of lynx1 confirmed a likely evolutionary link, and a rationale for why some animal toxins are so lethal in humans: they resemble something that already “fits” well in human neuronal receptors.
For the past two years, Ibanez-Tallon and her colleagues have been working with two kinds of animal venom peptides called bungarotoxin and conotoxin (from snakes and cone snails, respectively) to determine whether a natural peptide tether that holds its cousin, lynx1, in place, can be fashioned to reliably harness their activities. “We have generated 20 tethered toxins that block different receptors and ion channels,” explains Ibanez-Tallon.“We are now creating transgenic mice expressing some of these tethered toxins in specific neuronal circuits.The rationale of this approach is to block specific receptors in order to assess the role of these receptors in vivo.”
Though the more than five years of work that led up to an August 5 Neuron publication seems straightforward, electrophysiology experts the team consulted along the way were not convinced.“They cautioned that we wouldn’t be able to productively tether the toxins, because they’re so small that each amino acid is critical for their function, especially where Ines would have to join the peptide linker and lipid anchor,” says Heintz.“We considered those arguments carefully, and we also thought we’d try it despite the predictions.”
Not only does the tethering system work, Ibanez-Tallon demonstrated that it could be made to work for toxins with different functional characteristics. For example, in bungarotoxins, the lipid anchor needs only a short sequence to allow the toxin to bind to the receptor that chokes off the associated ion channel. However, in conotoxins, which are extremely small at 20 amino acids, the tether had to be quite long. “We thought it might not be possible to create a tether that would give conotoxins enough free rotation to get into the vestibule, or that the amino acid sequence of the longer tether might interfere with the activity of the toxin, but it works,” says Heintz.

October 15, 2004



 

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