|Institution:||University of Florida|
|Department:||Medical Sciences, Medicine|
|Full text PDF:||http://ufdc.ufl.edu/UFE0025079|
[3H]-DIHYDROERYSOVINE AS A MOLECULAR PROBE OF THE RESTING STATE ALPHA4BETA2 NICOTINIC ACETYLCHOLINE RECEPTOR The ?4?2 neuronal nicotinic acetylcholine receptor (nAChR) is the most abundant heteromeric nAChR in the mammalian brain and mediates much of the dopamine-releasing and cognition-enhancing effects of nicotine. Currently, many agonists and partial agonists at this nAChR are used as molecular probes or as drug candidates for treatment of certain neurodegenerative or neurodevelopmental diseases in which nAChRs are dysfunctional. In contrast, the ?4?2 antagonists have been less well studied and there is a need for more improving their nAChR selectivity. Coral bean (Erythrina) alkaloids have long been known for their curare-like effects on peripheral nAChRs. We have prepared a potent antagonist, dihydroerysovine (DHSOV), from the Erythrina alkaloid erysovine, and obtained a highly radioactive (tritiated) form of this compound to use as a molecular probe. DHSOV has a higher affinity at ?4?2 nAChR than the other known ?4?2 antagonists such as dihydro-?-erythroidine (DH?E) and erysodine. Using a saturation binding assay, I determined the affinities of DHSOV for nAChRs present in rat brain membrane (RBM) and in membranes from TSA-201 cells specifically expressing ?4?2 or ?4?5?2 receptors. The data suggested that the normally expressed ?4?2 receptor and another, as yet to be identified receptor have high affinity to DHSOV. My data also showed that DHSOV binding to the ?4?2 receptor is pH dependent at pH 7.4-9.0; affinity of binding decreased with an increase in pH. The ionized form of DHSOV seems to bind with high affinity at the ?4?2 receptor relative to the unionized form. In addition, the displacement of DHSOV binding in RBM by several common nAChR ligands indicates that [3H]-DHSOV is a useful molecular probe for measuring the affinity of an agonist for the ?4?2 receptor resting state.