Upregulation of nicotinic acetylcholine receptors (nAChRs) plays an integral role in nicotine addiction. Selectively promoting 1422 over other subtypes and more specifically the high sensitivity (HS) stoichiometry (2:3) over the low sensitivity (LS) stoichiometry (3:2), upregulation leads to an increased population of HS receptors at the membrane. This proposal investigates the mechanisms of membrane delivery and retrieval of 1422 receptors to determine the role trafficking and turnover play in nicotine induced upregulation. Additionally, nicotine-induced changes in the stoichiometry of these receptors will be investigated with single receptor resolution to directly visualize their role in upregulation. We will focus on how trafficking out of the endoplasmic reticulum (ER) varies with specific receptor subtypes and stoichiometries by observing variations in exocytosis and endocytosis activity of 1422. Observations of increased membrane dwell time in the presence of nicotine could primarily result from two distinct activities: binding of nicotine to the membrane receptor directly influences dwell time at the membrane or the two stoichiometries of 1422 have different dwell times and nicotine preferentially upregulates the stoichiometry with a longer lifetime giving the appearance of an increasing residence time at the membrane. The effects of nicotine on this behavior and how it induces differential trafficking to subcellular regions of mouse neuroblastoma cells and cultured neurons will be investigated using total internal reflection microscopy (TIRFM) and fluorescence resonance energy transfer (FRET) at both the single vesicle and single receptor level. Nicotine- induced redistribution of receptor ratios (HS vs. LS) clearly plays an important part in upregulation. Understanding how nicotine, in comparison to acetylcholine, cytisine, and DH2E, modifies 1422 trafficking to the membrane and residence time after insertion in different subcellular region will yield new insight into the mechanisms that drive upregulation. These studies will be the first to directly measure and visualize the two stoichiometries at the single molecule level. Characterization of nicotine-induced changes in nAChR trafficking and residence time at the membrane specifically correlated to stoichiometric identification in different subcellular regions will improve our understanding of addiction, facilitating the development of new and more effective smoking cessation treatments.
Tobacco consumption contributes to millions of cancer deaths every year and is also a leading factor in many other heart and lung diseases such as emphysema and vascular disease. Clearly developing methods to moderate smoking is a worldwide health imperative, and understanding the processes that lead to nicotine addiction will be crucial in the development of smoking cessation therapies. The studies here will focus on how nicotine affects receptor activity at the endoplasmic reticulum and the cell membrane particularly as it relates to receptor stoichiometries and subcellular localization.
|Srinivasan, Rahul; Richards, Christopher I; Xiao, Cheng et al. (2012) Pharmacological chaperoning of nicotinic acetylcholine receptors reduces the endoplasmic reticulum stress response. Mol Pharmacol 81:759-69|
|Richards, Christopher I; Srinivasan, Rahul; Xiao, Cheng et al. (2011) Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine. J Biol Chem 286:31241-9|