Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.
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abstract
We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few 44 nAChRs, but many 42 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the 4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of 44 receptor-containing vesicles with the PM by 2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2-3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of 44-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 M) did not alter the PM insertion frequency or trafficking behavior of 44-laden vesicles. In contrast, chronic nicotine substantially increased the number of 42-containing vesicle fusions at the PM; this stage in 42 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.
