In Eastern Australia resides one of the most notorious, poisonous members of the spider fauna: funnel-web spiders. With over forty different species and hundreds of toxins in their venom, these spiders cause serious medical injury or death to victims who are not treated with the effective antivenom. Would you want these spiders’ toxins anywhere near your skin?
At the outset, the response seems obvious, yet preliminary research performed by Michael Nitabach, Associate Professor of Cellular & Molecular Physiology and Associate Professor of Genetics, suggests that specific toxins may have the capabilities to inhibit the sensory function of pain transduction. Spearheading the project is Junhong Gui, a post-doctoral researcher in Nitabach’s laboratory at the Yale School of Medicine. Together, they are investigating basic nervous system function as a result of ion channel flow, using spider toxins as a manipulative tool to measure physiological responses.
Potential Medical Applications
Who would think to use poisonous toxins for potential medical applications? It turns out that the research project was not originally aimed towards human medicine. Nitabach has been using spider toxins as a tool for manipulating the nervous system in Drosophila, commonly known as fruit flies. He targets the nervous system’s ion channels, proteins that allow for the transference of ions across a cell membrane. Such ion channels regulate the flow of different ions and affect physiological procedures such as muscle contraction. “We have an interest in the biological function of a variety of different ion channel subunits, which have different specificities for different ions, and are regulated by different aspects of the physiological environment,” said Nitabach.
Nitabach and Gui went from conducting basic physiological manipulation to researching the possibility of an analgesic effect. The toxins in spider venom, called atracotoxins, come in hundreds of distinct varieties in the funnel-web spider. With such a large range of toxins, Gui and Nitabach thought that perhaps they could discover a few that have specific and potent activity that can inhibit mammalian ion channels involved in pain transduction.
Obstacles to Overcome
In conducting their research, Nitabach and Gui face a unique challenge: the spider and venom with which they work is neither naturally found in the United States nor legally transported into the country. Working with collaborators, they developed and implemented a technical platform for screening toxins that does not require native venom.
The key was receiving the sequence of the toxin’s amino acids which define its molecular identity by translation via messenger ribonucleic acid (mRNA). Armed with this genetic information, Gui can inject two types of synthetic mRNA into the oocyte (an early stage of the egg’s development) of an African clawed frog xenopus laevis. A single injection consists of the synthetic mRNA that encodes for a specific ion channel and the synthetic mRNA that encodes for one variety of toxin. The proteins are translated by the ribosome in the oocyte, expressing copies of several atracotoxins alongside a specific cloned ion channel in a closed system. The experiment is then repeated many times in different oocytes to screen all atracotoxins against the ion channel pain receptors.
Membrane-Tethering of the Toxins
A few subtle properties aid the procedure of developing and testing toxins against ion channels. Each toxin has a specified adjustment which makes it “membrane-tethered”—a process that ties the toxin to the surface of the oocyte co-expressing the ion channel. The co-injected oocyte thus sits in a culture dish, surrounded in medium, which keeps the frog egg alive. As a result, naturally secreted toxins will flow away into the culture medium rather than remain near the expressed ion channel. In the bulk medium, however, the concentration of toxin is too low to significantly affect the ion channel; thus, membrane-tethering is used to concentrate the secreted toxins around the expressed ion channel.
The biochemistry of the toxin allows for this synthetic membrane-tethering, a naturally occurring component of other cells in the nervous and immune systems. The toxin is secreted naturally but anchored to the outer membrane of the cell through glycolipid anchors. A glycolipid anchor is a chain of sugars, where one end of the chain forms a covalent link with the carboxyl-terminus of the secreted toxin protein. With this covalent link, lipid side chains are inserted into the outer plasma membrane of the cell to anchor the protein.
As a result of this synthesized addition of the glycolipid anchor, the atracotoxin is noted as a “chimeric fusion protein.” Coming from the classical Greek mythology of the Chimera monster composed of different body parts of a lioness, snake, and goat, chimeric proteins are composed of elements from various other naturally occurring proteins.
Physiological Testing
In order to determine the physiological effects of the toxin on the ion channel, Gui measures the current detected in the oocyte. Without toxin interference, ion channels transfer charged particles through the cell membrane, generating a net electric current. If the toxin blocks such a transmission of ions by binding to the ion channel, the ion channel undergoes a distortion that alters its 3D structure and thus changes how ions flow through it. Therefore, reduced or minimal current is measured. This method, Nitabach said, is a “well-established system for expressing cloned ion channels in isolation in a system where [one can] easily record biophysical properties of the channel in terms of the nature of the currents that flow through it.”
The technology behind this physiological testing is conveniently well established and commercially available to Nitabach’s lab. Using a physiological assay conducted with a type of ammeter that records the flow along the expressed ion channels with carefully positioned electrodes, Nitabach and Gui seek a preliminary candidate for activity against the channel. Nitabach explains: “If we could identify potent activity against ion channels of peripheral pain sensing terminals, even if the venom would kill…[it] could still be useful for certain types of peripheral pain systems when injected in limited quantities.”
Nitabach and Gui continue to emphasize that their work is still preliminary inquiry. While intriguing, the use of the atracotoxin as an agent to treat muscle spasms in pain or as a cosmetic product akin to Botox, is still a far distance into the future.
About the author: Robyn Shaffer is a sophomore in Berkeley College. She is the Production Manager for YSM and has worked in chemical and vascular surgery research. At this moment, she is wonderfully undecided about her major and other future plans.
Acknowledgments: The author would like to thank Professor Michael Nitabach and Junhong Gui, Ph.D. for their time and enthusiasm about their research.
Extra Reading:
“Cellular dissection of circadian peptide signals with genetically encoded membrane-tethered ligands.” Choi C, Fortin JP, McCarthy E, Oksman L, Kopin AS, Nitabach MN. Curr Biol. 2009 Jul 28;19(14):1167-75. Epub 2009 Jul 9.
“Electrical silencing of PDF neurons advances the phase of non-PDF clock neurons in Drosophila.” Wu Y, Cao G, Nitabach MN. J Biol Rhythms. 2008 Apr;23(2):117-28.
“Phase Coupling of a Circadian Neuropeptide With Rest/Activity Rhythms Detected Using a Membrane-Tethered Spider Toxin.” Ying Wu, Guan Cao¤, Beth Pavlicek, Xuan Luo, Michael N. Nitabach. PLoS Biology. November 2008.