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Show DETERMINATION OF ALPHA-COIMOTOX IN SPECIFICITY FOR BETA2 AND BETA4 SUBUNITS OF NICOTINIC ACETYLCHOLINE RECEPTORS David Shiembob, (Michael Mclntosh) Department of Biology, University of Utah Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels that are activated by nicotine and acetylcholine. The receptor is a heteromeric pentamer that is composed of alpha and beta subunits, of which there are 12 currently known neu- ronal subtypes. Different combinations of these subunits result in uniquely functioning subtypes of the receptor. nAChRs play a role in a number of biological functions such as pain, emotion, and auditory function as well as a number of disorders, such as Parkinson's and Alzheimer's disease. Alpha conotoxin Bul.3 is a relatively newly discovered neurotoxin that has been isolated from the venom of a predatory cone snail. This toxin acts on the nicotinic acetylcholine receptor in the brain. Its action largely depends upon what subtype of beta subunit it interacts with. The beta2 subunit displays a fast off rate of only a couple minutes whereas it takes hours for the toxin to come off of the beta4 subunit. This remarkable specificity makes it very useful as a probe to determine binding sites on the different beta subunits. The key residues have previously been found to lie in the extracellular portion of the receptor, and our task has been to fur-ther narrow down the binding region. We have done this by con-structing mutations that have one to four amino acids changed to those of the other subtype using the QuikChange Site Directed Mutagenesis Protocol. In addition to the mutations we have con-structed, we have been supplied by a collaborating lab with a number of chimeras and point mutations of the receptor to test. After constructing or receiving a chimera or point mutation, we transform it into E. coli. cells, grow up the cells, and extract the DNA. The DNA is then linearized, transcribed in vitro and the result-ing RNA is injected in Xenopus oocytes along with alpha3 subunit RNA, where it is expressed on the cell membrane as functioning receptors. We then use two electrode voltage clamp electrophysi- ology to assess the off rate of the toxin on the particular chimera or point mutation. In this manner we have been able to rule out a number of po-tential binding sites. We have also found that there are important residues in the first 54 amino acids of the receptor. In addition, we have recently discovered an amino acid at position 59 that appears to be crucial to the differing kinetics of the two subtypes. It appears that changing this single neutral amino acid to a basic one causes the beta2 receptor to behave as a beta4. This is an exciting development that we are still investigating. Faculty Sponsor Michael Mclntosh |