Serine residues in the &agr;4 nicotinic acetylcholine receptor subunit regulate surface &agr;4&bgr;2* receptor expression and clustering

Abstract

Background and purpose: Chronic nicotine exposure upregulates &agr;4&bgr;2* nicotinic acetylcholine receptors (nAChRs) in the brain. The goal of this study was to examine the role of three serine residues in the large cytoplasmic loop of the &agr;4 subunit on &agr;4&bgr;2* upregulation in neurons. Experimental approach: Serine residues S336, S470 and S530 in mouse &agr;4 were mutated to alanine and then re‐expressed in primary neurons from cortex, hippocampus and subcortex of &agr;4 KO mice. Mutant and wild type &agr;4 expressing neurons were treated with nicotine (0.1, 1 and 10 &mgr;M) and assessed for &agr;4&bgr;2* upregulation. Key results: &agr;4&bgr;2* nAChRs expressing S336A or S470A mutants were deficient at cell surface upregulation in both subcortex and hippocampal neurons. S530A &agr;4&bgr;2* mutants exhibited aberrant surface upregulation in subcortical neurons. None of the mutants affected surface upregulation in cortical neurons or upregulation of total &agr;4&bgr;2* binding sites in any region. Further, dense domains or clusters of &agr;4&bgr;2* nAChRs were observed in the neuronal surface. The impact of nicotine exposure on the intensity, area, and density of these clusters was dependent upon individual mutations. Conclusions and implications: Effects of &agr;4 nAChR mutants on surface upregulation varied among brain regions, suggesting that the cellular mechanism of &agr;4&bgr;2* upregulation is complex and involves cellular identity. We also report for the first time that &agr;4&bgr;2* nAChRs form clusters on the neuronal surface and that nicotine treatment alters the characteristics of the clusters in an &agr;4 mutant‐dependent manner. This finding adds a previously unknown layer of complexity to the effects of nicotine on &agr;4&bgr;2* expression and function. Graphical abstract Figure. No caption available.