Ivermectin Sensitive Glycine Receptor Mutations
Elizabeth Thornton interviewed by Marissa De Fratti
Q. How would you describe your project?
A. Well, I mutate plasmid (which is DNA) and then I put it into XL-1 Blue Supercompetent cells, which are a type of frog stem cells. After that we let them shake in the shakey-shakey machine overnight so that the DNA will be absorbed into the cells. Then we mini-prep the cells by putting them in different solutions (like ethanol) to lys them (which means to break them open) and then dump the solutions into test tubes. The test tubes are put into the centrifuge which rotates at high speeds and this process filters out unwanted material from the cell. Finally the solutions are sent with primers to Pittsburgh University for sequencing (which is the translation of the DNA to see if it was mutated).
Q. Why is any of that important?
A. The glycine receptor is directly related to nerves, including pain receptors. The glycine receptor is widely distributed throughout the body, mainly in the central nervous system. So, if the structure can be understood then we can understand why nerves keep sending signals to the pain center, even after the source of the pain has been healed. Hopefully, this will lead to the treatment of chronic pain.
Q. What do you hope to accomplish?
A. By mutating the cells we make them ivermectin (IVM) sensitive. This means that the glycine receptor will open when IVM attaches to it, when that happens we are able to control the glycine receptor state. The glycine receptor has an open, closed and desensitized state. When it’s open we can see the structure in that state. By looking at the receptor in its open state, which has never been seen before, we can then calculate what it would look like in its desensitized state. The desensitized state is almost the same as the closed state so with further calculations we can also determine its closed state.
Q. Has your project been successful so far?
A. Yes! We have all the mutations that we need: K206C, A290C, H419C. After the cells have been mutated, my part is over and the mutated glycine receptor plasmid is moved to the bacmid virus using Bac-to-Bac Baculovirus Expression System.