Neuronal nicotinic acetylcholine receptors (AChRs) exist in a variety of subtypes which play distinct roles in neuronal function in health and disease, and provide specific drug targets. AChRs are important in the pathological mechanisms of tobacco addiction and Parkinson's disease, and are potential drug targets in many diseases including Alzheimer's, schizophrenia, and ADHD. It is important to investigate the synthesis and functional properties of subtypes of human AChRs expressed in simple model systems in order to help explain their roles in complex genetically manipulated rodent nervous systems (e.g. knock out and knock in mice), and in humans. It is also important to develop systems in which highly selective drugs can be detected and their effects characterized. AChRs are formed from five homologous subunits organized around a central cation channel. Most AChRs are heteromeric, with two ACh binding sites formed by subunit pairs (e.g. a4?2) and a fifth """"""""accessory"""""""" subunit (e.g. ?2, ?3,a4, or a5) combined to form different AChR stoichiometries (e.g. (a4?2)2?2 or (a4?2) 2a4 or subtypes (e.g.(a4?2) 2a5). Our studies have revealed that accessory subunits influence efficiency of assembly, cation selectivity, as well as sensitivities to activation, desensitization, and allosteric modulators. Thus, it is especially important to investigate the roles of accessory subunits. Properties of a2 subunits are little known, but need investigation because they are more prominent in primates than rodents. AChRs containing a6 and ?3 subunits (among others, hence the designation a6* AChRs) are found almost exclusively in aminergic neuron endings where, for example, they modulate the release of dopamine. a6* AChRs are required for addiction to nicotine. They promote dopamine release from the same neurons that are lost in Parkinson's disease. They may also help mediate the neuroprotective effects of nicotine on these neurons, and may be important in ADHD and other diseases. a6 AChRs play an even larger role in dopamine release in primates than rodents. Thus, a6* AChRs offer excellent drug targets. Study of a6* AChR subtypes is important but difficult. Our preliminary results are encouraging. The use of linked subunits has allowed us to express a6* AChRs of defined subtypes, stoichiometries, and subunit orders. We have developed conditions for expressing a6* AChRs in transfected cell lines.
Aim 1 is to characterize alternate stoichiometries (e.g. (a4?2)2?2 and (a4?2)2 and alternate subtypes (e.g. (a4?2)2a5) of cloned human a4* and a2* AChRs expressed in pure forms.
Aim 2 is to efficiently express and functionally characterize cloned human a6* AChR subtypes. mAbs to the defined conformation dependent epitopes on the extracellular surface of a2, a6, ?3 and other subunits will be developed using novel AChR subunit/ACh binding protein chimeras as immunogens and will be used to study synthesis of these AChRs.
Nicotine acts through nicotinic receptors to cause addiction to tobacco, which results in more than 400,000 deaths/year in the United States. These receptors are found in the brain, peripheral nervous system and many non-neuronal tissues. Drugs directed at these receptors will help to reduce addiction to tobacco and may help treat Alzheimer's and Parkinson's disease, as well as many other neuronal and non-neuronal diseases. We have made a unique and growing collection of cell lines which express specific receptor subtypes. We also have a unique and growing library of monoclonal antibodies to the subunits of these receptors. Our studies will increase the understanding of how these receptors assemble and function normally and under the influence of nicotine and nicotinic drugs. The cell lines that we prepare can be used to detect and evaluate new drugs directed at these receptors.
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