Acta Pharmacologica Sinica (2009) 30: 828-841; doi: 10.1038/aps.2009.78

Acta Pharmacologica Sinica (2009) 30: 828-841; doi: 10.1038/aps.2009.78

Original Article

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Teresa A MURRAY^1,2,*, Qiang LIU¹, Paul WHITEAKER¹, Jie WU¹, Ronald J LUKAS¹

¹Barrow Neurological Institute, St Joseph’s Hospital and Medical Center, 350 W Thomas Rd, Phoenix, AZ 85013, USA; ²Center for BioOptical Nanotechnology, Biodesign Institute at Arizona State University, PO Box 5201, Tempe, AZ 85287-5201, USA

Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors.

Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC‑3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with ¹²⁵I‑labeled α-bungarotoxin (I‑Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.

Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC₅₀ values for the α7 nAChR-selective agonist, choline, and IC₅₀ values for the α7 nAChR-selective antagonist, methyllycaconitine. I‑Bgt binding was detected only after co-expression with hRIC‑3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs.

Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

Keywords: nicotinic receptor; fluorescent protein; confocal microscopy; protein engineering; protein expression; receptor trafficking; RIC-3; electrophysiology; dose-response relationship; radiotoxin binding

We wish to thank Dr Henry LESTER, California Institute of Technology, for the generous use of his facilities for performing confocal and TIRF studies. We also thank Dr Rigo PANTOJA and Dr Rahul SRINIVASAN for instruction and assistance using the TIRF and confocal microscope systems in Dr Lester’s lab. We also gratefully acknowledge Dr Sean MEGASON, California Institute of Technology for the kind gift of the pCS2‑mCherry construct; Dr Henry LESTER and Dr Raad NASHMI (formerly of the Lester lab and now at the University of Vancouver) for their gift of the α4‑HA‑YFP nAChR construct; and Dr Millet TREININ of Hebrew University for the gift of the hRIC‑3 construct through Dr William GREEN of the University of Chicago.

This work was supported by a National Institutes of Health Grant DA015389, the Barrow Neurological Foundation, and the Biodesign Institute at Arizona State University. TA MURRAY was supported by a National Science Foundation Graduate Research Fellowship and a Catholic Healthcare West SEED grant.

* Correspondence to Teresa A MURRAY, PhD.
Email biodesigner1@gmail.com
Received 2009-03-01 Accepted 2009-04-28

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