Selective and regulated trapping of nicotinic receptor weak base ligands and relevance to smoking cessation

Anitha P Govind,Yolanda F Vallejo,Jing-Zhi Yan,William N Green,Jacob R Stolz,Geoffrey T Swanson

doi:10.7554/elife.25651

Abstract

To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4β2-type nicotinic receptors (α4β2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing α4β2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of α4β2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing α4β2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in α4β2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed.

Highlights

Tobacco continues to be widely used world-wide, primarily via cigarette smoking, and is the leading cause of preventable deaths in the United States (National Center for Chronic Disease Prevention and Health Promotion (US) Office on Smoking and Health, 2014)
We determined if the smoking cessation drug and partial agonist varenicline, as well as other nicotinic acetylcholine receptors (nAChRs) ligands, caused upregulation of nAChRs or altered nicotine upregulation using HEK cells stably expressing a4b2type nicotinic receptors (a4b2Rs) or cortical neurons expressing endogenous a4b2Rs. a4b2R upregulation was assayed with two approaches: 125I-epibatidine binding (Figure 1) or patch-clamp recording of AChevoked currents (Figure 2)
We find that the smoking cessation agent varenicline (Chantix) has significant effects on a4b2Rs independent of its pharmacological action as a partial agonist

Summary

Introduction

Tobacco continues to be widely used world-wide, primarily via cigarette smoking, and is the leading cause of preventable deaths in the United States (National Center for Chronic Disease Prevention and Health Promotion (US) Office on Smoking and Health, 2014). The currently approved treatments for smoking cessation are nicotine replacement therapy, bupropion, and varenicline (Chantix). While varenicline is the most effective, successful quit rates only reach ~50% of smokers (Agboola et al, 2015). Nicotine binds to high-affinity nicotinic acetylcholine receptors (nAChRs) in the brain, and binding initiates its addictive effects. The a4b2 nAChR subtype (a4b2R) is closely linked to nicotine addiction (Govind et al, 2009; Vezina et al, 2007; Lewis and Picciotto, 2013). Loss of either subunit in a4 or b2 subunit knockout mice reduces the pharmacological and behavioral effects of nicotine (Picciotto et al, 1998; Marubio et al, 2003). Targeted expression of b2 subunits in the brain ventral

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