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 α4β4 nAChRs, but many α4β2 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 β4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4β4 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 α4β4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 μM) did not alter the PM insertion frequency or trafficking behavior of α4β4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4β2-containing vesicle fusions at the PM; this stage in α4β2 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.
Highlights
Nicotinic acetylcholine receptors4 are cation-selective transmembrane receptors expressed throughout the peripheral and central nervous systems [1, 2] and are assembled from various subunits (␣1–␣10, 2–4, ␥, ␦, and ⑀)
We used superecliptic phluorin (SEP) fused to the C terminus of ␣4 nicotinic receptor subunits to examine the subcellular distribution and trafficking dynamics of receptors expressed in cortical neurons
A, typical traces of ACh responses in a cell transfected with ␣4-enhanced green fluorescent protein (eGFP)2 subunits and a cell transfected with ␣4-SEP2 subunits
Summary
Nicotinic acetylcholine receptors (nAChRs)4 are cation-selective transmembrane receptors expressed throughout the peripheral and central nervous systems [1, 2] and are assembled from various subunits (␣1–␣10, 2–4, ␥, ␦, and ⑀). An epifluorescence image of a representative cortical neuron transfected with ␣4-SEP 4wt reveals fluorescence from all nonacidic compartments, i.e. from receptors at the membrane, in the ER, and in the cis-Golgi (Fig. 2A) (untransfected cells showed no detectable fluorescence).
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