Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12β1δε

Abstract

The human muscle-type nicotinic acetylcholine receptor α1₂β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development. Graphical abstract created with http://biorender.com.

Keywords: Molecular chaperones; Nicotine; Nicotinic acetylcholine receptor.

Graphical abstract created with http://biorender.com

Fig. 1

Plasmid maps of both transfer plasmids. Vector backbone is the construct of pL-SFFV.Reporter.RFP657.PAC (61395 Addgene) which was cloned via restriction sites AgeI and SpeI. a Insert for nAChR consists of a sequence of α1 subunit with His-tag, β1 subunit with HA-tag, SFFV promotor, δ subunit with myc-tag, ε subunit with flag-tag, and puromycin antibiotic resistance. b Insert for chaperones consists of a sequence of rapsyn with strep-tag-II, SFFV promotor, calnexin with rho1D4-tag, SFFV promotor, BiP with Ty1-tag, and blasticidin antibiotic resistance. Abbreviations of plasmid maps: 5′LTR 5′long terminal repeat, PBS primer binding site, RRE reverse response element, cppt central polypurine tract, SFFV splenic focus forming virus promotor, WPRE Woodchuck hepatitis virus posttranscriptional regulatory element, 3′LTR ∆U3 3′long terminal repeat deleted, AmpR ampicillin antibiotic resistance, pUC ori origin of replication (created with http://biorender.com)

Fig. 2

a Cell viability of transduced cell lines. Growth conditions with 9% FCS are shown in black and 16.5% FCS in gray. For comparison, untransduced (not TD CHO) cells were grown under the same conditions, and the viability of medium with 9% FCS was set to 100%. Three 1-TD cell lines with an empty backbone (RFP), nAChR, or 3 chaperones showed decreased viability. The viability of the 1-TD cell line with nAChR in comparison to the untransduced CHO cell line decreased significantly. The increased FCS concentration led to an increase in viability, especially in the case of 1-TD with nAChR which was significantly increased in comparison to the 1-TD nAChR CHO cell line grown with 9% FCS. Data are from three biological experiments each with eight technical replicates; the asterisk means significance with a p-value below 0.05. Light microscopy images of cell incubation on the same day: b untransduced CHO cells under incubating conditions with 9% FCS. c 1-TD nAChR cells under incubating conditions with 9% FCS in medium and d 1-TD nAChR cells under incubating conditions with 16.5% FCS in medium. Scale bars are 100 μm

Fig. 3

Western blot of transduced (nAChR) and untransduced CHO cell lanes. The detection of subunits was performed on separate blots. Lanes 1, 5, 9, and 12 show CHO cells without transduction for α1 (1), β1 (5), δ (9), and ε (12). Lanes 2, 4, 8, and 11 show CHO cells with nAChR after transduction (1-TD) for α1 (2), β1 (4), δ (8), and ε (11). Lanes 3, 6, 7, and 10 show the marker, 50- and 70-kDa bands are indicated

Fig. 4

In-cell Western of CHO cell lines transduced with one-target approach (1-TD). Target proteins were detected by their specific tag and fluorescence signals were normalized to 30,000 cells. The black bar is the empty backbone vector with the reporter RFP from 1-TD RFP. Dark gray bars are the three chaperones with their tags strep-tag-II, rho1D4, and Ty1 from 1-TD 3 chaperones. Light gray bars are the subunits of nAChR with their tags His, HA, myc, and flag from 1-TD nAChR. Data are from three biological experiments with nine technical replicates each.

Fig. 5

Representative whole-cell currents in 1-TD nAChR and not TD CHO cells. The black current corresponds to untransduced CHO (not TD CHO) and gray current to nAChR (1-TD nAChR) cells. The traces show the current response to 15 μL of 70 μM nicotine applied at a rate of 114 μL/s. The black horizontal bar indicates the exposure time. Currents were elicited at a holding potential of −70 mV

Fig. 6

Cell viability of 1-TD nAChR cell line under hypothermic conditions. The black bar indicates untransduced (not TD CHO) cells which were grown at 37 °C and 9% FCS and their viability was set to 100%. 1-TD with nAChR at 37 °C and 16.5% FCS (light gray) showed a viability of about 88%. Viability of 1-TD with nAChR at 34 °C for 7 days and 16.5% FCS (dark gray) is minimally decreased. Viability of 1-TD with nAChR at 31 °C for 48 h and 16.5% FCS (white) is significantly decreased in comparison with 1-TD nAChR at 37 °C. Data are from three biological experiments each with eight technical replicates, * means significance with p-value below 0.05.

Fig. 7

On-cell Western of CHO cells transduced as one target (1-TD nAChR). Target proteins were detected by their specific tag in native conditions and fluorescence signals were normalized to 30,000 cells. The housekeeper protein integrin (black bar) was used as a reference. Gray bars are the subunits of nAChR with their tags His, -HA, -myc, and -flag from 1-TD without incubation of nicotine. White bars are the subunits of nAChR with their tags His, -HA, -myc, and -flag from 1-TD with incubation of 30 μM nicotine for 24 h. Signals are from three biological experiments with six technical replicates