Desensitization is an intrinsic property of many neurotransmitter receptors, characterized by a decline in response to maintained activation by neurotransmitters and by changes in affinity to agonists and a variety of drugs. Although the phenomenon of desensitization has long bene known, its significance in normal and disordered brain function, as well as in learning and memory is still unknown. The nicotinic acetylcholine receptor (AChR) has been used widely to study desensitization, and it is known that phosphorylation at a specific site on a particular subunit is critical, or whether an increase in overall level of phosphorylation is more important in modulating the AChR desensitization. Evidence also suggests that Ca2+ ions modulate the AChR desensitization. The goal of this research is to understand the mechanisms involved in desensitization. To pursue this goal, I will use the AChR as a model system. The initial aim is to identify phosphorylation sits on the particular receptor subunits which are responsible for determining the rate of desensitization. The studies will be made by eliminating potential phosphorylation sites on the AChR at the DNA level, and then expressing site directed mutant AChRs in Xenopus oocytes. The large size of these cells facilitate many procedures including microinjection, voltage-clamp recording of ACh-activated currents, and manual isolation of the surface membrane. The interaction of Ca2+ with the phosphate groups and negatively charged amino acids, introduced at phosphorylation sites by site directed mutagenesis, is the AChRs in oocytes will also be studies by internal injection of EGTA or Ca2+. Subsequently, I will focus on the identification and characterization of the regions of the alpha, gamma and epsilon subunits of AChRs involved in controlling the rate of desensitization. Extrajunctional AChRs of mammalian muscles expressed in oocytes appear to desensitize more slowly than Torpedo AChRs, also synthesized in oocytes. This appears to be a consequence of the structural differences in the gamma subunits of the respective AChRs. Similarly, junctional muscle AChRs expressed in Xenopus oocytes, in which the gamma subunit is replaced by the epsilon subunit, desensitize more rapidly than extrajunctional AChRs. Furthermore, the hybrid AChR consisting of the muscle alpha and Torpedo beta, gamma and delta subunits desensitizes much faster than the normal Torpedo AChR. By contrast the hybrid AChR formed by the Torpedo alpha and muscle, beta, gamma and delta subunits desensitizes more slowly than the normal muscle AChR. Therefore, the alpha, gamma and epsilon subunit cDNAs will be very important tools to study the mechanisms involved in desensitization, chimeric alpha, gamma or epsilon subunit cDNAs with different combinations of Torpedo and muscle counterparts will be made and expressed in oocytes.
