Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

Tamara Darsow,T.K Booker,Juan Carlos Piña-Crespo,Stephen F Heinemann

doi:10.1074/jbc.m501157200

Tamara Darsow, T.K Booker + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m501157200

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Abstract

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed alpha(4)beta(2) nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested.

Highlights

Neuronal nicotinic acetylcholine receptors1 in mammalian brain compose a family of proteins encoded by 11 genes (␣2–7, ␣9–10, and ␤2–4) that assemble into pentameric ligandgated ion channels [1,2,3,4]
We find that transport of nicotinic acetylcholine receptor (nAChR) through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors
We show that in this system, chronic nicotine treatment induces the up-regulation of surface ␣4␤2 nAChRs in a manner that is similar to nAChR up-regulation observed in the brains of human smokers and in animal models chronically exposed to nicotine

Summary

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) in mammalian brain compose a family of proteins encoded by 11 genes (␣2–7, ␣9–10, and ␤2–4) that assemble into pentameric ligandgated ion channels [1,2,3,4]. Up-regulation is observed in the brains of human smokers [13] as well as in chronically nicotine-treated animal models [8, 14] and in cultured cells heterologously expressing nAChRs [15,16,17,18], suggesting that up-regulation requires basic, conserved cellular processes. Mechanisms dictating the number of surface receptors at the plasma membrane may be involved in nicotine-induced up-regulation. To directly test this hypothesis, we analyzed the up-regulation and trafficking of surface ␣4␤2 nAChRs using a cell culture model that expresses functional surface-localized ␣4␤2 receptors. Our analysis of nicotine-dependent nAChR trafficking suggests that up-regulation is regulated not via modulation of endocytic trafficking but at a biosynthetic step prior to insertion of ␣4␤2 nAChRs into the plasma membrane

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