Andon Placzek - US grants

DESCRIPTION (provided by applicant): Amino acid residues in the second transmembrane (TM2) domain of nicotinic acetylcholine receptors (nAChRs) have been shown to have important regulatory functions in studies using site-directed mutagenesis and heterologous expression systems. This is true for sites in the beta subunit TM2 domain of both muscle and high-affinity neuronal nAChRs, as well as homopentameric alpha7 receptors. This proposal outlines a course of experimental study that will attempt to identify the possible roles of homologous sites in the alpha7 TM2 domain that have previously been established in the beta subunit as critical determinants of receptor function and pharmacology. This involves the substitution of amino acids from the muscle and neuronal beta subunit TM2 domains for identical residues in the alpha7 TM2 domain. Pharmacological characterization will include sensitivity to activation by agonist, sensitivity to non-competitive inhibitors (NCIs), and the degree of residual inhibition or desensitization. Functional characterization will involve analysis of receptor kinetics and divalent ion permeability. Thus far, the preliminary findings indicate that mutation of the alpha7 TM2 6' position to include either the muscle Beta1 subunit sequence or the neuronal Beta2/Beta4 subunit sequence, dramatically changes the response kinetics of the mutant receptors so that the decay rate of macroscopic currents is more like that of the respective wild-type beta subunit containing receptor than wild-type alpha7. The research plan outlined here will serve to further the training of the applicant in molecular biology and electrophysiology, and will help to identify the potential contribution of specific sites within the alpha7 TM2 domain to important aspects of its function and pharmacology.

[unreadable] DESCRIPTION (provided by applicant): There is evidence for both long and short-term changes in synaptic function in several areas of the brain known to mediate reward signals. One type of change occurs in dopamine (DA) neurons of the midbrain. It has been shown that a single, acute exposure to nicotine produces a shift in the subtypes of ionotropic glutamate receptors mediating evoked synaptic transmission. This shift is manifested as an increase in the AMPA/NMDA receptor ratio. This increase is thought to be related to the insertion of postsynaptic AMPA-type receptors that is known to underlie long-term potentiation (LTP). Preliminary data show that in young (21 - 35 postnatal day) C57 mice, a single intraperitoneal injection of nicotine causes an increase in the AMPA/NMDA receptor ratio of evoked glutamate currents in putative midbrain DA neurons. The same effect is also seen in older (60 - 90 postnatal day) mice but with a rightward shift in the dose-response relationship. This suggests that while nicotine is still able to produce the kind of synaptic plasticity that is commonly associated with cellular models of learning and memory (i.e., LTP), the potency of the effect is decreased with age. It is predicted that in younger animals, this type of plasticity will be more sensitive to lower doses of nicotine, and that this relative sensitivity may contribute to the increased vulnerability of adolescents to nicotine addiction. Furthermore, this proposal outlines a course of experiments to determine the effects of exposure to nicotine during this critical developmental period, and to determine if prior exposure to nicotine at a young age lowers the dose threshold for nicotine induced plasticity. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Smoking-related diseases are a major cause of preventable death in the U.S. and around the world. Despite the wealth of information regarding the health risks of tobacco use, many continue to smoke, attesting to the addictive power of nicotine. Research designed to illuminate the mechanisms of nicotine addiction may help in the development of effective methods of prevention and treatment of what is ultimately a life-threatening behavior. [unreadable] [unreadable] [unreadable]