Bernhard M , Laube B .

	Figure 1. Purification and functional characterization of the α1-His GlyR in SMA-copolymer nanodiscs. (a) Schematic representation of GlyR (PDB: 3JAE) in native nanodiscs (upper) and chemical structure of SMA-copolymer (lower) with a ratio of n:m of 2:1 used in this study. Size exclusion chromatogram (b) and SDS-PAGE analysis (c) showing an efficient separation of α1-His GlyR nanodiscs. Peak fraction (*) shows a clear band (black arrow) between 40 and 55 kDa, corresponding to the α1 GlyR (MW: 48 kDa) and a band migrating at ~ 10 kDa corresponding to SMA copolymer. Gel image was cropped, indicated by a grey cropping line.Subfigure (a) was created using Abobe Illustrator CC version 24.3 (https://www.adobe.com/kr/products/illustrator.html).
	Figure 2. Functional characterization of SMA copolymer solubilized α1-His GlyR. (a) Example trace of primary thermophoresis data. Thermophoretic movement of α1-His GlyR nanodiscs is expressed as the change in fluorescence signal between initial fluorescence Fcold (0 s) and fluorescence after thermodiffusion Fhot (15 s) and was calculated as ratio of both values as described in the “Materials and Methods” section. Inset shows an amplification of representative fluorescence traces of the thermophoretic movement of the fluorescence labeled α1-His GlyR between 14 and 15 s (Fhot) obtained at different glycine concentrations [0 and 1 (black), 10 and 100 (gray) and 1000 and 3000 µM glycine (red)]. (b) The change in thermophoretic movement upon binding of increasing concentrations of Gly results in a change of the relative fluorescence between the unbound state (black) and glycine-bound state (red) after 15 s. (c) Initial fluorescence count distribution for each concentration is under 10% and showing no ligand-dependent fluorescence quenching. (d) Dose–response curve obtained from MST experiments of α1-His GlyR. Binding of glycine to fluorescence-labeled α1-GlyR was obtained with a titration series from 3 mM to 0.73 µM in PBS buffer, pH 7.4. The change in thermophoretic signal leads to a cEC50 of 65 ± 22.8 µM. Error bars represent SEM between n = 3 independent experiments.
	Figure 3. Functional analysis of α1-GFP GlyR nanodiscs from HEK293 cells and X. laevis oocytes. (a) Dose–response relationship of α1-GFP GlyR obtained by MST experiments and electrophysiological recordings from HEK293 cells and oocytes. MST data points were inversely normalized for better comparison with dose–response curves obtained from electrophysiological measurements. Data are shown in mean ± SEM. (b) Western blot of SMA-copolymer solubilized GFP-GlyR α1 obtained from the membrane fractions of oocytes and HEK293 cells, show a single band at the calculated molecular weight below 70 kDa. Western blot image was cropped, indicated by a grey cropping line. (c) Electrophysiological experiments obtained from oocytes and HEK293 cells revealed aEC50 values 212.9 ± 21 µM and 68.8 ± 7.4 µM, respectively. cEC50 values of 52.6 ± 40.8 µM (n = 4) and 40.9 ± 13.4 µM (n = 4) obtained from oocytes and HEK293 cells showing no significant difference (p = 0.41). The aEC50 obtained from is significantly higher (p < 0.01) than the measured cEC50, while the aEC50 and cEC50 obtained from HEK293 cells show no difference (p = 0.23). Error bars represent SEM between independent experiments. (d) Signal amplitudes obtained from MST experiments reveal no differences between HEK293 cells (signal amplitude = 4.04 ± 1.09) and oocytes (signal amplitude = 3.38 ± 1.09; p = 0.41, n = 4). Data are shown in mean ± SD. Unpaired two-side t test for statistics.
	Figure 4. Binding characteristics of the partial agonist taurine to α1-GFP GlyR nanodiscs. (a) Dose–response data for glycine and taurine of heterologous expressed α1 GlyR from X. laevis oocytes. Taurine acts as a partial agonist with an aEC50 value of 843 ± 16 µM reaching a maximum current of 61% compared to glycine (n = 3). Taurine currents are normalized to the maximum glycine currents for each cell. Dose–response data of glycine are the same as shown in Fig. 3. Error bars represent SEM. (b) MST binding experiment of α1-GFP GlyR with a taurine titration series of 6 µM to 12.5 mM results in a cEC50 value of 473.8 ± 46.1 µM (n = 3). Error bars represent SEM. (c) Exemplary α1-GFP GlyR MST data of taurine (blue circles) and glycine (black circles) obtained from oocytes displaying a difference in their maximal thermophoretic mobility (grey and blue arrows). (d) Comparison of the signal amplitudes of α1-GFP GlyR SMALPs expressed in HEK293 cells and oocytes for glycine and taurine. Binding of taurine leads to a significant decreased thermophoretic movement (p = 0.024, unpaired two-side t test, n = 3) with signal amplitudes of 1.32 ± 0.14 compared to glycine-bound receptors with signal amplitudes of 3.38 ± 1.09. Data are shown in mean ± SD.
	Figure 5. Schematic model of the activation mechanism of GlyRs for full and partial agonists. (a) The general activation mechanism of GlyRs includes at least three conformational states, whereby two conformations adopting a contracted ligand-bound ECD. Binding of a full agonist (red dot) induced an ECD closure of the resting receptor, while the ion channel is still shut (intermediate state). This ECD closure finally opens the ion channel and activates the receptor (open state). The ability of an agonist to change the conformation within the ECD (cEC50) was measured by MST, while the general ability to activate the receptor (aEC50) was determined by electrophysiological methods. (b) Binding of a partial agonist (purple) initiate an incomplete closure of the ECD (1). The receptor is either activated by a further ECD closure (2, grey) that leads to an ion channel opening (open state) or can directly open with a less contracted ECD (2, black). (c) GlyR activation in HEK293 cells and oocytes. Glycine binding to GlyRs in HEK293 cells is characterized by a contraction of the ECD and a rapid channel opening, reflected by similar cEC50 and aEC50 values. Binding of glycine to GlyRs in oocytes is also characterized by an efficient reorientation of the ECD with an impaired channel opening, possibly stabilizing an intermediate state with a contracted ECD and a closed channel pore.Figure was drawn using Abobe Illustrator CC version 24.3 (https://www.adobe.com/kr/products/illustrator.html).

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