Bachmann M et al. (2021), Efficient expression of a cnidarian peptide-gat...

XB-ART-57799

Channels (Austin) 2021 Dec 01;151:273-283. doi: 10.1080/19336950.2021.1882762.

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Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells.

Bachmann M , Ortega-Ramírez A , Leisle L , Gründer S .

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Hydra Na+ channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the Hydra nervous system. They have previously been successfully characterized in Xenopus oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca2+ imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.

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	Figure 1. Functional expression of HyNaC2/3/5 in COS-7 and HEK 293 cells. Top, representative currents elicited by applying 10 µM RFamide I (black bar) to COS-7 cells (left) or HEK cells (right) expressing HyNaC2/3/5; both cells had similar capacitances (~20 pF). Bottom, current densities for COS and HEK cells (mean ± SD); n = 12 for each cell line; p < 0.0001
	Figure 2. Western blot analysis of native and codon-optimized HyNaCs. A) Western blot representative for three independent experiments. Cells had been co-transfected with HyNaC2, HyNaC3 and HyNaC5; one of the subunits carried an HA-tag as indicated. Note that for optimized HyNaCs samples had been diluted 100-fold. B) Densitometric analysis of bands in the range of 50–75 kDa quantifies the increased expression of codon-optimized HyNaCs. AUC, area under the curve
	Figure 3. Electrophysiological characterization of codon-optimized HyNaC2/3/5 in HEK 293 cells. A) Top, representative current traces of native (gray trace; current elicited by 10 µM RFamide I) and codon-optimized (black trace; current elicited by 1 µM RFamide-II) HyNaC2/3/5 expressed in COS-7 cells (left) or HEK cells (right); all cells had similar capacitances (~20 pF). Bottom, current densities of native and codon-optimized HyNaC2/3/5 (mean ± SD). n ≥ 10 for each cell line; ****, p < 0.0001 (unpaired t-test). Data for native HyNaC expressed in COS-7 cells are from Figure 1. B) Top, representative currents of codon-optimized HyNaCs expressed in COS-7 cells elicited by 10 µM RFamide I or 1 µM RFamide II. Bottom, current densities after activation with the two peptides (mean ± SD). n = 10, p = 0.87 (unpaired t-test). C) Left, representative currents of codon-optimized HyNaC2/3/5 elicited by different concentrations of RFamide II. Right, concentration–response curve. Error bars represent S.D. The line represents a fit to the Hill equation. n = 8
	Figure 4. Ca2+ responses of HEK 293 cells expressing native and codon-optimized HyNaC2/3/5. A) Mean Ca2+ responses of 10 cells to stimulation with 1 µM Hydra-RFamide II (RFa II) or 1 µM ionomycin. Responses are presented as ratio of 340/380 nm fura-2 fluorescence; basal fluorescence was subtracted. Dotted lines represent the SD. B) Quantification of 6 experiments similar to the one shown in A from 2 different transfections (mean ± SD; ***, p < 0.001, unpaired t-test). For individual cells, the mean of the two Ca2+ responses to stimulation with Hydra-RFamide II was normalized to the response to ionomycin. The total number of cells analyzed was n = 36 for native HyNaCs and n = 48 for codon-optimized HyNaCs
	Figure 5. Effect of the HA-tag on HyNaC current density. Current densities of native HyNaC2/3/5 (white bar), codon-optimized HyNaC2/3/5 (gray bar) and codon-optimized HyNaC2/3/5 with one or more subunits carrying an HA-tag (green, yellow, pink and purple bars). Error bars represent S.E.M; n = 7 to 15; *** p < 0.0001 (one-way ANOVA)
	Figure 6. Western blot analysis of glycosylation of codon-optimized HyNaCs. A) Linear diagrams of HyNaC2, HyNaC3, and HyNaC5 illustrating the predicted positions of N-glycans, transmembrane domains M1 and M2, and the C-terminal HA epitopes. The one conserved glycan is marked in red. B) Treatment with PNGase F or Endo H reduced the molecular mass of all HyNaC subunits, indicating N-glycosylation. Stars indicate the endo-H resistant forms. Cells had been co-transfected with HyNaC2, HyNaC3 and HyNaC5; one of the subunits carried an HA-tag as indicated. Western blot is representative for three independent experiments

References [+] :

Assmann, The comprehensive analysis of DEG/ENaC subunits in Hydra reveals a large variety of peptide-gated channels, potentially involved in neuromuscular transmission. 2014, Pubmed, Xenbase