Vasseur L et al. (2019), Importance of the Choice of a Recombinant Syste...

XB-ART-56562

Int J Mol Sci 2019 Jun 28;2013:. doi: 10.3390/ijms20133181.

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Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG.

Vasseur L , Cens T , Wagner R , Saint N , Kugler V , Chavanieu A , Ouvry C , Dupré C , Ferry G , Boutin JA .

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Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.

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Species referenced: Xenopus
Genes referenced: kcnh2
GO keywords: potassium channel activity

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	Figure 1. Voltage activation and pharmacological properties of hERG(S1-coil) recorded in Xenopus oocytes. (A). Current-voltage relationship for hERG-wt and hERG(S1-coil). Currents measured at the end of a 4 seconds long depolarizing pulse (from âˆ’70 to 30 mV) were normalized to the maximal current for hERG-wt (n = 21) and hERG(S1-coil) (n = 19). (B). Steady-state voltage-dependence of activation. The tail currents at âˆ’50 mV were normalized to the peak tail current, plotted against the amplitude of the depolarizing pulse and fitted with a Boltzmann function to estimate the potential for half-activation (V1/2) and the slope value (k) for hERG-wt (V1/2 = âˆ’33.3 Â± 0.9 mV, k = 7.5 Â± 0.4 mV, n = 21) and hERG(S1-coil) (V1/2 = âˆ’27.7 Â± 2.3 mV, k = 6.0 Â± 0.4 mV, n = 19). Note that the values obtained with hERG(S1-coil) are significantly different from those obtained with hERG-wt (p < 0.01). (C). Inhibition curves for hERG-wt (n = 9) and for hERG(S1-coil) (n = 13) by E-4031 determined by measuring the tail current at âˆ’40 mV in the presence of increasing concentration of E-4031 and normalized to the control current measured in the absence of drug at a voltage of âˆ’40 mV after 2 seconds of depolarization at +20 mV. Curves are fitted with a logistic function. (D). Inhibition curves for hERG-wt (n = 7) and for hERG(S1-coil) (n = 8) by the BeKm-1. hERG wild type = empty circles; hERG(S1-coil) = filled squares.
	Figure 2. Characterization of the binding properties of [3H]-dofetilide to hERG(S1-coil) expressed in three recombinant systems. The chimeric hERG(S1-coil) channel was expressed in the yeast P. pastoris, Sf9 insect and mammalian HEK293 cells (transient transfection and stable and inducible cell lines). Membranes were prepared for each system. Saturation ligand binding experiments with [3H] dofetilide were performed using 10 Âµg of total membrane protein. Specific binding was calculated from total and non-specific measurements in triplicate (unspecific values are comprised between 0.5 and 1 pmol/mg). All error bars are all less than 0.6 pmol/mg. Empty triangles = P. pastoris; Filled circles = HEK stable; Filled triangles = Sf9; Empty circles = HEK transient.
	Figure 3. Quantification of hERG in P. pastoris and HEK membranes. A. hERG(S1-coil) expressed in P. pastoris was solubilized in 1% dodecylphosphocholine (DPC) and purified on a TALONÂ® resin. The presence and purity of the protein was validated by SDS-PAGE (lane 1) and Western blot (lane 2). The purified protein was quantified by the BCA test. B. Increasing quantities of purified proteins and membrane preparations of P. pastoris and HEK were studied by Western blot using an anti-hERG antibody. C. hERG can be quantified in membranes of P. pastoris (triangle) and HEK (square) cells after fitting the linear equation between the amount of purified hERG(S1-coil) from A used as a standard and the fluorescent area on Western blot (RÂ² = 0.9986).
	Figure 4. Detergent extraction of hERG from the membranes of three recombinant systems. Membranes were prepared from yeast P. pastoris, Sf9 insect cells or stable mammalian HEK cells expressing hERG(S1-coil). A sample of each membrane containing 20 Âµg of total protein for P. pastoris and HEK cells or 1000 Âµg of protein for Sf9 cells was incubated with 1% dodecylmaltoside (DDM) or 1% DPC. Solubilized (S) and non-solubilized (NS) fractions were studied by Western blot with an anti-His antibody.
	Figure 5. hERG channel function in lipid bilayers after HEK expression and DDM solubilization. A. The purified hERG(S1-coil) was overexpressed in stable HEK cell line, solubilized in 1% DDM and the resulting supernatant was purified on a Strep affinity resin and eluted with 2 mM biotin. B. hERG(S1-coil) multichannel currents recorded at an applied potential of +100, 0 and âˆ’100 mV. B. Single hERG(S1-coil) channel currents recorded at an applied potential of âˆ’100 mV. C. Current trace and related point-amplitude histogram.

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