Localization of Plasma Membrane and Intracellular Neuronal Nicotinic Acetylcholine Receptors Using Quantitative Imaging in Mammalian Cells

Summary

This protocol describes expression and quantification of neuronal nicotinic acetylcholine receptors subunits using the mammalian Neuro-2a cell line. pH-sensitive fluorescent tags and confocal microscopy enable precise evaluation of receptor localization at the plasma membrane versus those expressed in pH-neutral intracellular compartments, facilitating the study of nAChR trafficking and pharmacological modulation.

Abstract

Quantification of neuronal nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane of mammalian cells is important for the development of novel target-selective therapeutics and for studying chaperone proteins that modulate receptor trafficking. This study presents a robust set of methodologies to express both homomeric and heteromeric nAChRs in mammalian Neuro2a (N2a) cells and to quantify their surface versus intracellular localization using pH-sensitive fluorescent tags. pHuji and superecliptic pHluorin (SEP) are pH-sensitive proteins that can be engineered to tag recombinant proteins, allowing visualization of the spatial distribution of nAChR subunits. These fluorescent tags emit at neutral pH but are quenched in acidic environments. To track the relative internal versus plasma membrane localization of α7, α4, and β2 nAChR subunits, either pHuji or SEP is attached to the C-terminus of each DNA construct. To express α7-pHuji together with its chaperone NACHO, or (α4-SEP; β2-pHuji) in N2a cells, a lipid-based DNA transfection reagent is used. The transfection reagent and plasmid DNA are incubated separately in reduced-serum medium at room temperature, then combined to form lipid-DNA complexes that facilitate plasmid delivery into cells. Following incubation, cells are imaged using live-cell confocal microscopy under both high- and low-pH conditions or in the presence of a subtype-selective fluorescent ligand. Images are analyzed using corrected total cell fluorescence in the relevant fluorophore channels, providing quantitative information on the distribution of fluorescently tagged nAChR subunits between the plasma membrane and intracellular compartments. Live-cell confocal imaging enables real-time tracking of nAChR subunit localization to distinguish differences between internal and surface receptor populations. Robust plasma membrane expression of α7-pHuji or (α4-SEP; β2-pHuji) is achieved within 24 h at 37 °C. This method can be adapted to include additional chaperones, auxiliary subunits, or fluorescent ligands for the study of nAChR trafficking, pharmacological modulation, and disease-related alterations in nAChR expression.

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated transmembrane ion channels expressed on the plasma membrane of many cell types, including neurons, glial cells, and immune cells. These receptors are composed of α (α2-α10) or β (β2-β4) subunits that assemble to form homomeric or heteromeric subtypes, with α7 and α4β2 nAChRs being the most abundant within the brain^1,2. Dysregulation of these subtypes has been associated with Alzheimer's disease, nicotine use disorder, sleep-related hypermotor epilepsy, Parkinson's disease, and major depress....

Protocol

NOTE: The following procedure describes methods that have been used successfully to transiently transfect the N2a cell line with nAChR subunits (Figure 1). Robust plasma membrane expression is achieved within 24 h, as captured with live-cell confocal microscopy.

Figure 1: Schemat....

Results

Following the above transfection procedures, α7 nAChRs and the NACHO chaperone protein were co-expressed in N2a cells. Both non-transfected and transfected cells were labeled with αBTX-AF647 24 h post-DNA transfection. Because αBTX is not membrane-permeable, all fluorescence detected originates from receptors present on the plasma membrane. In Figure 2A,A′, weak αBTX-AF647 staining was observed in non-transfected N2a cells, as expected. N2a cells endogenously express a small.......

Discussion

The methodologies presented for expressing and quantifying nAChR subunits in mammalian N2a cells address long-standing challenges in achieving rapid (~24 h) and abundant nAChR (> 60%) plasma membrane expression for drug discovery, localization, and trafficking studies. A key strength of this set of protocols is its adaptability for both homomeric (α7) and heteromeric (α4β2) nAChRs, and it is likely applicable to other nAChR subtypes, along with the co-expression of chaperone proteins. By leveraging pH-.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA (1X)	Gibco	25200-056	Cell detachment reagent
Alpha-Bungarotoxin Alexa Fluor 647	Invitrogen	B35450	Alpha-Bungarotoxin conjugate
BSL-level 1 Biosafety Cabinet	Nuaire	NU-425-400	Sterile cell culture working area
Calcium chloride	Aventor/VWR	BDH9224	Buffer component
D-(+)-Glucose	MilliPore Sigma	G8270	Buffer component
EMEM with L-glutamine	ATCC	30-2003	Cell medium
Endotoxin-free DNA	Made in house using Zymo kits	not applicable	nAChR DNAs to be used for cell transient transfections
Endotoxin-free microcentrifuge tubes, 0.5 mL	Aventor/VWR	89000-010	DNA and transfection preparation
Endotoxin-free microcentrifuge tubes, 1.5 mL	Aventor/VWR	20170-038	DNA and transfection preparation
Fetal Bovine Serum	Aventor/VWR	97068-085	Serum supplement
Fluoview FV10i	Olympus	FV10i	Confocal Microscope
Glass Bottom MatTek Dishes	MatTek	P35GC-0-10-C	Imaging dishes
GraphPad Prism v. 10	GraphPad Prism	https://www.graphpad.com/features	Data analysis software
HEPES	MilliPore Sigma	H3375	Buffer component
Lipofectamine 2000	Invitrogen	11668019	Transfection reagent
Magnesium chloride hexahydrate	Thermo scientific	413410025	Buffer component
MES hydrate	MilliPore Sigma	M5287	Buffer component
mTagBFP2-pBAD	AddGene	54572	Control DNA to be used for cell transient transfection
Neuro 2 a (N2a) cells	American Type Culture Collection	CCL-131	Cells used for DNA transfections and nAChR expression
Opti-MEM	Gibco	31985-070	Reduced serum medium
Penicillin/Streptomycin	Aventor/VWR	K952-100mL	Antibiotic
Peristalsis pump	Gilson	MiniPuls3	Buffer changes when imaging
Phosphate-buffered saline	Quality Biological	119-069-131	Saline buffer
Pipet Aid	Drummond	140084	Precise liquid transfer
Pipet tips (sterile and endotoxin-free, 1000uL)	Rainin	30389216	Precise liquid transfer
Pipet tips (sterile and endotoxin-free, 20uL)	Rainin	30389229	Precise liquid transfer
Pipet tips (sterile and endotoxin-free, 250uL )	Rainin	30389246	Precise liquid transfer
Pipettes, P10	Rainin	L-10XLS	Precise liquid transfer
Pipettes, P100	Rainin	L-100XLS	Precise liquid transfer
Pipettes, P1000	Rainin	L-1000XLS	Precise liquid transfer
Potassium chloride	MilliPore Sigma	P3911	Buffer component
Serological pipets (sterile and endotoxin-free, 10 ML )	Aventor/VWR	89130-898	Precise liquid transfer
Serological pipets (sterile and endotoxin-free, 25 mL )	Aventor/VWR	89130-900	Precise liquid transfer
Serological pipets (sterile and endotoxin-free, 5 mL )	Aventor/VWR	76201-710	Precise liquid transfer
Serological pipets (sterile and endotoxin-free, 50 mL )	Aventor/VWR	75816-088	Precise liquid transfer
Sodium chloride	Aventor/VWR	BDH9286	Buffer component
Tissue Culture Flask	Falcon	353109	Growing dishes
ZymoPure II Plasmid Maxiprep Kit	ZYMO RESEARCH	D4203-A + D4203-B	Production of endotoxin-free DNA
α4-SEP	ThermoFisher Scientific GeneArt Gene Synthesis Services	https://www.thermofisher.com/us/en/home/life-science/cloning/c-misc/geneart.html	Custom gene synthesis services
α7-pHuji	Twist Bioscience	https://www.twistbioscience.com/	Custom gene synthesis services
β2-pHuji	ThermoFisher Scientific GeneArt Gene Synthesis Services	https://www.thermofisher.com/us/en/home/life-science/cloning/c-misc/geneart.html	Custom gene synthesis services