Modulation of Recombinant Human Î±1 Glycine Receptors by Mono

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Modulation of recombinant human #1 glycine receptors by mono-and disaccharides – a kinetic study Ulrike Breitinger, Heinrich Sticht, and Hans-Georg Breitinger ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00044 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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ACS Chemical Neuroscience

Modulation of recombinant human α1 glycine receptors by mono- and disaccharides – a kinetic study

Ulrike Breitinger*1, Heinrich Sticht2 and Hans-Georg Breitinger*1

1

Department of Biochemistry, The German University in Cairo,

Main Entrance of Al Tagamoa Al Khames, New Cairo 11835, Egypt. 2

Department of Bioinformatics, Institute for Biochemistry,

Friedrich-Alexander-Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen

*To whom correspondence should be addressed: Ulrike Breitinger Hans-Georg Breitinger Department of Biochemistry The German University in Cairo email: [email protected] , [email protected] Tel.: +20-2-2759-0699 FAX: +20-2-2758-1041

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Abstract Glycine receptors (GlyRs) mediate fast synaptic inhibition in spinal cord, brainstem, and higher brain centers. Recently, glucose was identified as a positive modulator of GlyRmediated currents. Here, we investigated extent and kinetics of the positive modulation of recombinant human α1 glycine receptors by different mono- and disaccharides and sorbitol using patch-clamp recording techniques. Glucose and fructose augmented glycine-mediated whole-cell currents with an EC50 of 6-7 mM. At concentrations >10 mM, the maximum of current enhancement was reached within ~30 min. Kinetics of GlyR modulation resemble those of protein glycation. On-rates were 24 h). Galactose, the C4-epimer of glucose and the sugar alcohol sorbitol had no effect on GlyR currents. Recent cryoelectron microscopy structures were used to identify a potential binding site for saccharides near the ivermectin binding pocket with lysine 143 as possible attachment site. The GlyR mutant α1(K143A) was not potentiated by glucose, suggesting an involvement of this residue in glycine receptor modulation by saccharides.

Keywords Ligand-gated ion channels; glycine receptor; glucose; saccharides; protein modulation by saccharides; glycation


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Introduction The inhibitory glycine receptor is one of the principal mediators of rapid synaptic inhibition in the mammalian spinal cord and brain stem (1). Glycine receptors are pentameric ion channels that belong to the cys-loop family of ligand-gated ion channel receptors. To date, four ligandbinding alpha subunits, α1-4, and one structural β- subunit are known, of which α4 is not expressed in humans. Each GlyR subunit contains an N-terminal extracellular domain, four transmembrane (TM) domains, as well as a large intracellular TM3-TM4 loop which is responsible for intracellular modulation (1-3). Under physiological conditions, the GlyR channel is primarily selective for chloride, leading to postsynaptic hyperpolarization (1-3). GlyR-mediated signalling is involved in the regulation of muscle tone and movement, as well as signal transmission in the human retina (4), cochlea (5), and hippocampus (6, 7). Furthermore, transmission through spinal cord α3 receptors has been shown to be an integral part of pain signalling (8-10). Several loss-of-function mutations in GlyR genes are known to underly motor disorders, such as human hyperekplexia (STHE, startle disease, OMIM 149400), stiff man syndrome, bovine myoclonus and other neuromuscular diseases (11-13). Modulation of glycine receptor-mediated transmembrane currents by alcohols and anaesthetics, which act as potentiators of the receptor, were described in detail (12, 14). Recently, cryo-electron microscopy structures of the zebrafish GlyR in complex with glycine, strychnine, and ivermectin (15), and strychnine-bound α3 glycine receptors (16) were reported, identifying the primary sites for agonist and antagonist binding.

Recently, glucose and fructose were shown to be positive modulators of inhibitory α1 and α1/β glycine receptors (17). When added to the growth medium, glycine-mediated currents were augmented, leading to a ~2.5- fold decrease of EC50. Glucose primarily reduced the large variability of GlyR responses that had been characterized previously (18, 19). When 3 ACS Paragon Plus Environment


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glucose was added to the culture medium overnight, potentiation was even stronger, producing a decrease of EC50 values up to 4- fold (17). Similar potentiation of GlyR-mediated currents was observed for recombinant human α3 glycine receptors (20). Glucose-mediated potentiation of glycine receptor activity showed an EC50 < 10 mM, i.e. at physiologically relevant concentrations, and suggests a role of glucose as endogenous modulator of glycinergic transmission (17, 20). One possible mechanism of protein modification by sugars is glycation, the covalent attachment of sugar moieties to lysine residues of target proteins. It is an extracellular, non-enzyme-catalyzed process which leads to the formation of primary adducts, which eventually convert to of advanced glycation endproducts, including protein crosslinks, that are implicated in numerous complications of diabetes and aging (21).

Here, we investigated the activity of a series of mono- and disaccharides, as well as the sugar alcohol sorbitol as glycine receptor potentiators. Concentration dependence and time course of GlyR modulation by the saccharides were determined. Glucose, fructose and mannose were most active, with on-kinetics for GlyR modulation < 30 min, and very slow off-kinetics (>24 h). Modulation by disaccharides was similar in extent, but of slower time course. Surprisingly, even at the highest concentration of 50 mM galactose, the C4-epimer of glucose, and the sugar alcohol sorbitol were both inactive. The GlyR mutant α1(K143A), where a potential glycation site is removed, was not potentiated by sugars, indicating an involvement of this residue in glucose modulation.


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Results and Discussion Glucose has been shown to potentiate glycine evoked currents on α1, α1/β, and α3 glycine receptors (17, 20). Here, we investigated the effects of glucose-related monosaccharides glucose, fructose, galactose and mannose, as well as the disaccharides sucrose and lactose, and the sugar alcohol sorbitol as modulators of α1 glycine receptors. Human homomeric α1 glycine receptors were recombinantly expressed in HEK293 cells and the EC50 for receptor channel activation by glycine was determined using whole-cell patch-clamp recording techniques. Under all conditions, cells gave stable responses to application of glycine (Figure 1). Throughout, HEK293 cells were cultured in standard medium (MEM Eagle, supplied with antibiotics and 10 % FBS). This medium contains 1 g/l of glucose, corresponding to 5.5 mM, and was designated as MEM5.5. Glucose is an essential nutrient, so we did not attempt cell culture or receptor expression in glucose-free medium. This allowed us to avoid unspecific changes in cell viability and compare our results to published data where cells are routinely maintained in media containing 1 g/l of glucose.

Two lines of experiments were followed, culturing receptor-expressing cells in standard culture medium (5.5 mM glucose), and varying the concentration of saccharides in the extracellular perfusion buffer (Exp 1), and preincubation of GlyR-expressing cells in media containing varying amounts of saccharides, followed by patch-clamp measurements in saccharide-free extracellular buffers (Exp 2). When saccharides were added to the recording medium (Exp 1), effects on GlyR-mediated currents that developed on a time scale of ~ 10 – 120 minutes could be observed. Preincubation experiments (Exp 2) explored the time scale of 1 - 16 hours of exposure to saccharides. Glucose was added to the growth medium resulting in an end concentration of 5.5 + x mM glucose. For pretreatment experiments involving other sugars, the concentration of each saccharide was added to the base value of 5.5 mM glucose



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present in control medium (see Methods). The concentration of each sugar and the Total Saccharide Concentration in mM (TSC) present in the medium is noted throughout the text.

Direct modulation of GlyR responses by bath application of saccharides Recombinant wildtype α1 glycine receptors expressed in HEK293 cells gave stable wholecell current responses to glycine stimulation (Figure 1). When glucose, fructose and sorbitol were directly added to the recording buffer (Exp 1), increased sugar concentrations reduced EC50 values of glycine from 39.3 ± 2.7 µM to 15.5 ± 2.6 µM for 50 mM glucose and 12.8 ± 2.0 µM for 50 mM fructose (Figure 3A, Table 1). Maximum current Imax was not affected by saccharides in the bath, while changes in EC50 were significant (p ≤ 0.05). When 50 mM sorbitol was added to the bathing solution the EC50 value of glycine was slightly reduced to 28.4 ± 6.8 µM, but the change was not significant compared to control (p > 0.1, Figure 2G, Table 1). Likely, sorbitol does fit poorly or not at all into the glucose binding pocket, a similar conformational selectivity of saccharide binding has been reported for the GLUT1 glucose transporter (22).

Pre-treatment of recombinant GlyRs with monosaccharides In pre-treatment experiments (Figures 2, 3, Table 2), addition of glucose for ~ 16 hours resulted in a reduction of glycine EC50 values that occurred over a very narrow range of glucose concentration. At 5.5 mM glucose (control medium), we determined an EC50 of glycine of 39.3 ± 2.7 µM. Glycine EC50 values were determined after 16 hours of preincubation using additional 1.0 mM (TSC 6.5), 2.0 mM (TSC 7.5), 4.5 mM (TSC 10), 14.5 mM (TSC 20) and 45.5 mM (TSC 50) glucose in the growth medium and gave average EC50

values

of

24.9 ± 1.8 µM,

11.4 ± 1.4 µM,

14.0 ± 1.0 µM,

12.1 ± 1.3 µM, and

12.8 ±1.2 µM, respectively (Figures 2A, 3C, Table 2). Here, minimal glycine EC50 values 6 ACS Paragon Plus Environment

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were observed at 7.5 mM total glucose. When studying shorter preincubation of 1 hour, 7.5 mM total glucose resulted in a reduced but not minimum glycine EC50 value of 27.3 ± 1.9 µM (Figure 3C, Table 2) while exposure to 10 mM total glucose (TSC 10, EC50 = 15.7 ± 3.6 µM, Figure 2A, 3C, Table 2) produced the maximum reduction of glycine EC50. All observed shifts of EC50 were statistically significant, (One-way ANOVA, p ≤ 0.05). The minimum of EC50 that we observed varied between 11 µM and 14 µM. A strong cell-to-cell variability of GlyR responses has been reported before (18, 19). Notably, the spread of EC50 values was reduced, but not completely eliminated in presence of sugars, as previously reported (17).

Fructose and Mannose produced augmentation of glycine-mediated currents similar to glucose (Figure 2B-C, 3D-E). Fructose reduced the glycine EC50 value to 8.6 ± 0.5 µM (Table 2, p < 0.05), even lower than observed with glucose (EC50 = 12.8 ± 1.2 µM, p < 0.05). For mannose, the final EC50 value was 12.5 ± 0.5 µM (Table 2, p < 0.05). Thus, the structurally related monosaccharides glucose, fructose (corresponding ketose), and mannose (C2-epimer of glucose) all were positive modulators of the glycine receptor. Surprisingly, galactose, the C4-epimer of glucose, did not modulate GlyR responses. Even after 16 hour preincubation with 50 mM of galactose (TSC 55.5), EC50 was 35.4 ± 3.2 µM, not significantly different from control (39.3 ± 2.7 µM, p > 0.05, Figure 2F, 3F, Table 2). Sorbitol, which had shown small and non-significant reduction in glycine EC50 in bath application (Exp 1) was also inactive as GlyR modulator under pretreatment (Exp 2) conditions. After preincubation with 50 mM sorbitol (TSC 55.5) for 1, 4, and 16 hours, EC50 values of 33.2 ± 3.0 µM, 34.8 ± 7.4 µM and 34.4 ± 3.7 µM, respectively, were determined (Figure 2G, 3B), all differences from control (39.3 ± 2.7 µM) were not significant (p > 0.05).



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The monosaccharides glucose, fructose, and mannose were all positive modulators of recombinant glycine receptors, showing similar potency for the reduction of EC50 values. Sorbitol was inactive, which could be rationalized from its conformation. It also lacks the aldehyde function, which is required for covalent interaction with proteins (21, 23). The finding that galactose, the C4-epimer of glucose, was not able to modulate GlyR currents, was unexpected, especially since galactose is known to interact with proteins even faster than glucose (24, 25). Our results suggest that the sugar binding site on the glycine receptor may not accommodate galactose. Similar effects were reported for the sodium-dependent glucose transporter whose affinity for the transport across the plasma membrane is much higher for glucose than for galactose (26). Also, GLUT7, recombinantly expressed in Xenopus oocytes, shows high-affinity transport for glucose and fructose, while galactose, 2-deoxy-D-glucose, and xylose are not transported (27). Thus, although a glycating agent, galactose likely does not bind to the glycine receptor effectively. The absence of any modulatory effects with sorbitol and galactose is consistent with a specific modulation of the glycine receptor by saccharides and argues against unspecific effects due to changes in osmolarity or viscosity of the recording buffers.

Pre-treatment – disaccharides Preincubation with sucrose and lactose also induced an augmentation of glycine evoked currents (Figure 2D-E, 3G-H), although on a slower time scale than that of monosaccharides. After one hour of pretreatment with 50 mM (TSC 55.5) of sucrose or lactose, EC50 of glycine was only slightly decreased (30.1 ± 4.2 µM for sucrose, 29.3 ± 7.4 µM for lactose, p > 0.1). The maximum of GlyR modulation was observed after 4 hours of pretreatment using 50 mM (TSC 55.5) of disaccharides, EC50 values then were 15.2 ± 0.7 µM, and 16.4 ± 4.6 µM for sucrose and lactose, respectively (Table 2). These values were not further reduced after 16 hours of incubation (Figure 2D-E, 3G-H, Table 2), where glycine EC50 values were 16.8 ± 8 ACS Paragon Plus Environment

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1.7 µM (sucrose) and 16.1 ± 1.2 µM (lactose). Disaccharides were effective modulators, but reduction of glycine EC50 was slightly less than with monosaccharides (sucrose/ lactose ~ 16 µM, monosccharides ~11 µM). Although statistically significant (p < 0.05), it is likely that these small differences are due to natural variation and not physiologically relevant.

Kinetics of GlyR modulation by saccharides Modulation of GlyR responses by saccharides was measured on a time scale between 1 and 16 hours (Figure 4). At concentrations ≥ 10 mM of total glucose (TSC 10), augmentation was at its maximum within one hour, and no differences in glycine EC50 values between preincubation times of 1 hour and 16 hours were found (Figure 4A), consistent with an onrate of < 1 h. At small concentrations (additional 1-2 mM of glucose, TSC 6.5 and TSC 7.5, respectively), glucose modulation was slower and not complete after 1 hour (EC50 = 27.3 ± 1.9 µM at 7.5 mM glucose, TSC 7.5) compared to overnight treatment (EC50 = 11.4 ± 1.4 µM). For 6.5 mM (TSC 6.5) and 7.5 mM (TSC 7.5) of total glucose, time constants for reduction of EC50 were 2.3 ± 1.4 h and 1.77 ± 0.57 h, respectively (Figure 4A). For 10 mM, 20 mM and 50 mM total glucose in the growth medium (TSC 10, TSC 20, TSC 50, respectively), we determined time constants of 0.37 ± 0.22 h, 0.46 ± 0.11 h and 0.33 ± 0.17 h, respectively (Figure 4A, Table 3). The time course of GlyR potentiation by fructose and mannose was similar to that of glucose. Time constants were found to be 2.0 ± 1.0 h for 1 mM additional fructose (TSC 6.5), and 0.39 ± 0.06 h, 0.56 ± 0.09 h, and 0.44 ± 0.09 h for 10 mM (TSC 15.5), 20 mM (TSC 25.5) and 50 mM (TSC 55.5) of fructose, respectively (Figure 4A, Table 3). For mannose the time constants were 1.57 ± 1.04 h at 2 mM (TSC 7.5), 0.41 ± 0.16 h at 10 mM (TSC15.5) and 0.42 ± 0.07 h at 50 mM (TSC 55.5, Figure 4A, Table 3). Glycine receptor modulation by disaccharides was ~ 5-fold slower. For concentrations of 50 mM of additional disaccharide (TSC 55.5) we determined time constants of 1.63 ± 0.73 h for sucrose 9 ACS Paragon Plus Environment


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and 1.64 ± 0.37 h for lactose (Figure 4C, Table 3), both significantly higher than that of glucose (0.33 ± 0.17 h). This difference could be due to slower binding to the receptor protein. Disaccharides may be hydrolyzed to give monosaccharides, although at pH 7.4 this effect is likely negligible. While association kinetics were on the order of hours, off-kinetics were considerably slower. GlyR-transfected HEK293 cells were first exposed to 50 mM of glucose (TSC 50) or sucrose (TSC 55.5), then the medium was exchanged and cells kept in control medium (TSC 5.5) for another 24 hours before EC50 was measured (Figure 4D). Since cell viability was affected by consecutive changes of sugar in the medium, off-kinetics could be followed for 24 hours only. In this set of experiments, EC50 for glycine changed from the control value (39.3 ± 2.7 µM) to 13.4 ± 1.3 µM after 16-h preincubation with 50 mM glucose (TSC 50). After 24 hours of recovery, EC50 had increased to 17.8 ± 1.0 µM. For sucrose, EC50 was 16.8 ± 1.7 µM after 16h preincubation in 50 mM sucrose (TSC 55.5), and 21.7 ± 2.3 µM after a further 24 hours in control medium. The extent of recovery was not yet statistically significant (p > 0.05, oneway ANOVA), but it clearly indicated a return towards control values. The data are consistent with much slower off-kinetics (τ >> 24 h) than on-kinetics for the modulation of glycine receptor function by sugars. It is noted that the off-kinetics we observed are on a time scale on which receptor turnover, i.e. internalization of surface receptors and delivery of new receptors to the plasma membrane, does also take place. Likely, the off-rates we found represent both, loss of sugar and receptor turnover. While both processes cannot be distinguished by our observations, it is obvious that on-rates for saccharide modulation are faster than the combined rates of receptor turnover and saccharide removal.


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Effective concentrations of different saccharides as GlyR modulators The concentration-dependence of modulation of glycine EC50 values by glucose (Figure 4B, Table 3) gave an EC50 value for glucose as positive GlyR modulator of 6.38 ± 0.09 mM (TSC 6.38) equal to 0.88 ± 0.09 mM of additional glucose (added to the base concentration of 5.5 mM, i.e. TSC 5.5). The EC50 value of GlyR modulation was 0.85 ± 0.24 mM (TSC 6.35) for fructose, and 0.78 ± 0.07 mM (TSC 6.28) for mannose (Figure 4B-C, Table 3), both very similar to that of glucose (0.88 ± 0.09 mM). For disaccharides, EC50 values for GlyR modulation were 0.69 ± 0.09 mM (TSC 6.19) for sucrose and 0.63 ± 0.17 mM (TSC 6.13) for lactose. Thus, all saccharides showed a similar effective concentration of GlyR modulation. Notably, all sugars that were capable of positive modulation had the same effective EC50, i.e. GlyR modulation by 10 mM of glucose (5.5 mM + 4. 5 mM, TSC 10) was similar to the modulation by 5.5 mM of glucose + 4.5 mM of fructose (TSC 10). This is consistent with the assumption that all sugars exert their effects in an additive manner, targeting the same site on the receptor.

Glycation as a possible mechanism for GlyR modulation It is noted that time course and effective concentration range for modulation of glycine receptors by saccharides are closely similar to those of protein glycation. The positive modulation of GlyR responses upon bath application of glucose and fructose (Exp 1) could be mediated through non-covalent interactions. In contrast, potentiation of GlyR responses upon pre-exposure to sugars, followed by EC50 determination in sugar-free recording buffers, is consistent with a covalent modification of the receptor protein by saccharides, although very tight non-covalent binding, however unlikely, cannot be excluded. A study of non-enzymatic glycation of hemoglobin (23) suggests a reaction scheme where glucose (G) and hemoglobin (H) form an aldimine intermediate (H=G) which slowly converts to the stable ketoamine (HG), with a time course similar to the one observed here. 11 ACS Paragon Plus Environment


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H + G

k1

k2

H=G

HG

k-1 Here, k1 = 0.3x10-3 mM-1h-1, k-1 = 0.33 h-1, k2 = 0.0055 h-1 (scheme and time constants from (23)). Fructose and glucose are both effective agents for protein glycation, with fructose reported to glycate even faster than glucose (28). Disaccharides are active, but on a slower time scale than monosaccharides (29). The reactivities of saccharides for receptor modulation that we observed in this study match their ability to glycate, and are consistent with protein glycation as possible mechanism underlying GlyR augmentation.

Glycation of haemoglobin is well-documented, especially in the treatment of diabetes, and is strongly affected in the clinically relevant range between 5 and 15 mM of glucose (24, 25, 3034). All saccharides that were able to potentiate GlyR responses were active in this concentration range. Raising glucose from 5.5 mM to 6.5 mM would already produce a significant reduction of glycine EC50 of the glycine receptor and if the total concentration of saccharides reaches 10 mM, the maximum of GlyR potentiation is reached. In healthy humans, resting blood glucose levels are around 5.5 mM (80 – 100 mg/dl). In untreated diabetic patients, resting blood glucose may reach 11 mM (200 mg/dl), and even higher in severe cases. Fasting blood glucose levels of 7 mM and higher are considered indicative of diabetes (http://www.diabetes.co.uk). Especially in cases of diabetes and other diseases where blood glucose levels escape control, glycine receptor modulation through elevated glucose might be considered in clinical evaluations. The range of sugar concentration for GlyR modulation is thus narrow and precisely in the clinically and physiologically relevant range.


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A putative glucose binding site Our observations revealed two requirements for the positive modulation of glycine receptors by saccharides, (i) presence of an aldehyde function that can undergo formation of early glycation products, (ii) conformation of the modulating saccharide that allows effective interaction with the receptor. We thus searched for a possible saccharide binding site on the glycine receptor, which was able to accommodate a sugar moiety, and would allow covalent attachment, which in case of protein glycation is directed at lysine residues. The binding site for ivermectin does match these requirements. Ivermectin is a macrocyclic lactone that acts as allosteric modulator of GlyRs. It binds at the transmembrane interface of two adjacent receptor subunits (15), interacting with hydrophobic residues in membrane helices α3 of the first subunit and α1 of the second subunit (Figure 5D, left panel). Indeed, a potential glycation site is present in this binding pocket, namely lysine residue α1(K143). This residue is implicated in channel gating (35), and receptor surface expression (36). According to a recently published cryo-EM structure of the glycine/ivermectin-bound glycine receptor, residue α1(K143) is located close to the binding site for ivermectin (15). Molecular modeling revealed that the upper part of the ivermectin binding pocket could readily accommodate a linear glucose molecule (Figure 5D, right panel). In the model, the aldehyde group of glucose and the side chain –NH2 group of lysine would be positioned close to each other, suggesting that residue K143 may indeed be glycated. This assumption was confirmed by a prediction routine (37) that identified K143 as a possible glycation site. In order to test this hypothesis, we removed the glycation site in position α1(K143). Indeed, the glycine receptor mutant α1(K143A) was insensitive to glucose modulation. The mutant had a twofold increased EC50 value for glycine (79.6 ± 2.5 µM) compared to the wildtype (EC50 = 39.3 ± 2.7 µM). After preincubation with 50 mM glucose, EC50 of the α1(K143A) mutant was 78.7 ± 3.7 µM (Figure 5B, D-E, Table 2). Thus, glucose had no detectable influence on this mutant, 13 ACS Paragon Plus Environment


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consistent with an involvement of residue α1(K143) in the modulation of glycine receptor function by saccharides. In previous experiments (17), we have shown that receptor activation by ivermectin is not affected by the absence or presence of glucose. Neither an unspecific effect of the K143A mutation on GlyR activation and gating, nor the existence of other glycation sites on the receptor cannot be ruled out from our experiments. Nevertheless, homomeric a1 glycine receptors, as used here, have five identical ivermectin binding sites, so glucose and ivermectin may both bind to the same receptor complex, and presence of one compound would not prevent the other from binding. Indeed, results from structure modelling and experiment agreed well and suggest position α1(K143) as a likely glycation site on the receptor protein.

Overall, the present study shows that increased amounts of glucose and related saccharides can lead to a positive modulation of glycine receptor responses. Kinetics, concentration dependence, and structure modelling suggest glycation of the GlyR protein as a likely mechanism for such a modulation. Thus, the concentration of glucose and other sugars in culture medium and recording buffers are variables that need to be controlled in laboratory experiments. In clinical situations that involve elevated levels of blood glucose, glycation and modulation of glycine receptors may be considered for diagnose and therapy.


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Methods Site-directed Mutagenesis – Single nucleotide exchanges corresponding to the mutation GlyR α1(K143A) were introduced by PCR-mediated site-directed mutagenesis using an overlap extension PCR approach. Mutagenesis primers (Biomers, Ulm, Germany) contained nucleotides specific for the amino acid exchange. PCRs were set up following the manufacturer’s instructions using high-fidelity Taq

polymerase

(Roche Molecular

Biochemicals, Mannheim, Germany). The final fragments were cut with restriction enzymes and reinserted into the pRK5 vector. The mutated clone was sequenced across the PCRgenerated sequence to verify successful mutagenesis (LGC, Berlin, Germany).

Cell Culture and Transfection – HEK293 cells were grown in 10 cm tissue culture Petri dishes in MEM (Sigma, Deisenhofen, Germany) supplemented with 10 % FBS (Invitrogen, Karlsruhe, Germany) and 5000 i.u. Penicillin/Streptomycin at 5 % CO2 and 37 °C in a water saturated atmosphere. For experiments, cells were plated on poly-L-lysine treated glass coverslips in 6 cm dishes. Transfection was performed 1 day after cell passage using Gen-Carrier (Epoch Lifesciences, Sugarland, TX, USA): 1.3 µg of receptor DNA, 1.3 µg of green fluorescence protein DNA and 2.6 µl GenCarrier were used, following the manufacturer’s instructions. Measurements were performed 2 to 4 days after transfection. For pre-treatment experiments, saccharides were added 1 to 2 days after transfection. Exposure to saccharides was maintained for the indicated time until patch-clamp recordings were performed.

Electrophysiological Recordings and Data Analysis – Current responses from GlyRtransfected HEK293 cells were measured at room temperature (21°C – 23°C) at a holding potential of -50 mV. Whole-cell recordings were performed using a HEKA EPC10 amplifier (HEKA Electronics, Lambrecht, Germany) controlled by Pulse software (HEKA Electronics). 15 ACS Paragon Plus Environment


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Recording pipettes were pulled from borosilicate glass (World Precision Instruments, Berlin, Germany) using a Sutter P-97 horizontal puller (Sutter, Novato, CA). Solutions were applied using an Octaflow system (NPI electronics, Tamm, Germany), where cells were bathed in a laminar flow of buffer, giving a time resolution for solution exchange and re-equilibration of about 100 ms. The external buffer consisted of 135 mM NaCl, 5.5 mM KCl, 2 mM CaCl2, 1.0 mM MgCl2, and 10 mM Hepes (pH adjusted to 7.4 with NaOH); the internal buffer was 140 mM CsCl, 1.0 mM CaCl2, 2.0 mM MgCl2, 5.0 mM EGTA, and 10 mM Hepes (pH adjusted to 7.2 with CsOH). Saccharides (Sigma-Aldrich, Deisenhofen, Germany) were added to the growth medium or the bath solution as indicated. Concentration-response data were fitted to the Hill equation

I glycine I sat

=

[Glycine]nHill using a nonlinear algorithm in nHill [Glycine]nHill + EC50

Microcal Origin (Additive, Friedrichsdorf, Germany). Here, Iglycine is the current amplitude at a given glycine concentration, Isat is the maximum current amplitude at saturating concentrations of glycine, EC50 is the glycine concentration at half-maximal current responses, and nHill is the Hill coefficient. Currents from each individual cell were normalized to the maximum response at saturating glycine concentrations. Significance of differences between EC50 values were determined using one-way ANOVA with p ≤ 0.05 (*) and p ≤ 0.01 (**) taken as significant. Whole-cell recordings were either taken in glucose-free extracellular buffer, where GlyR-transfected HEK293 cells had been exposed to sugar in the culture medium for 1-16 hours, or cells were grown in control medium (5.5 mM glucose), and 50 mM sugar were added to the extracellular buffer during electrophysiological experiments as indicated. In all experiments EC50 values were determined for each individual cell from a non-linear fit of concentration-response data to the logistic equation (above). An un-weighted average was calculated from all individual EC50 values, without considering the fitting errors. All data are given as means ± standard error of the means.


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For analysis of on-kinetics of saccharide modulation, cells were preincubated in 1 - 50 mM of saccharide for indicated times (0 h = control, 1 h, 4 h, 16 h), and then EC50 determined in sugar-free recording buffers as described above. From a plot of glycine EC50 versus preincubation time, the kinetic constants for modulation by saccharides were determined from a non-linear fit (Microcal Origin, Additive, Friedrichsdorf, Germany) of the data to a monoexponential decay function EC50 (t ) = EC50 (0) * e

−

t

τ

+ EndEC50 , where EC50(t) is the glycine

EC50 value after t hours of modulation, EC50(0) is the control value (no saccharide modulation), t is the time constant of modulation, and EndEC50 is the fitted glycine EC50 value at t = ∞. The standard medium for cell culture contained 1g/l (5.5 mM) of glucose. This medium (TSC 5.5) was used as control. Since glucose is an essential nutrient, whose absence would damage cells, no experiments in glucose-free medium were performed and saccharides were always added to control medium as indicated. For glucose, unless noted differently, total concentration (5.5 mM plus additional amount) is given. For other saccharides, individual concentrations are given, and additional 5.5 mM glucose were present in the medium. Throughout the text, the total amount of saccharides (TSC) in the culture medium is indicated.

Structure Modelling – Sequence-based prediction of glycation (36) sites was performed using the NetGlycate site (http://www.cbs.dtu.dk/services/NetGlycate-1.0.). Modelling of the glucose-bound GlyR was performed based on the cryo-EM structure of ivermectin-bound GlyR (PDB code: 3JA F; (15)). The coordinates for the linear form of glucose were adapted from the MalL crystal structure (PDB code 4M56; (38)). The glucose modeled into the upper part of the ivermectin binding pocket using UCSF Chimera (39) and the K143 sidechain was rotated to minimize the distance to the ligand. RasMol (40) was used for structure analysis and visualization.



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Acknowledgements We thank Prof. Dr. Cord-Michael Becker for a kind gift of glycine receptor cDNA, and Eileen Socher for help with the model of glucose-bound GlyR. Expert technical assistance by Mousa Abdalla Mousa is gratefully acknowledged. The authors report no conflict of interest.


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References 1.

Breitinger, H.-G. (2014). Glycine Receptors, Volume doi: 10.1002/9780470015902.a0000236.pub2 (Chichester: eLS. John Wiley & Sons Ltd).

2.

Dutertre, S., Becker, C.M., and Betz, H. (2012). Inhibitory glycine receptors: an update. J Biol Chem 287, 40216-40223.

3.

Lynch, J.W. (2009). Native glycine receptor subtypes and their physiological roles. Neuropharmacology 56, 303-309.

4.

Wässle, H., Heinze, L., Ivanova, E., Majumdar, S., Weiss, J., Harvey, R.J., and Haverkamp, S. (2009). Glycinergic transmission in the Mammalian retina. Front Mol Neurosci 2, 6.

5.

Dlugaiczyk, J., Singer, W., Schick, B., Iro, H., Becker, K., Becker, C.M., Zimmermann, U., Rohbock, K., and Knipper, M. (2008). Expression of glycine receptors and gephyrin in the rat cochlea. Histochem Cell Biol 129, 513-523.

6.

Xu, T.L., and Gong, N. (2010). Glycine and glycine receptor signaling in hippocampal neurons: diversity, function and regulation. Prog Neurobiol 91, 349-361.

7.

Eichler, S.A., Forstera, B., Smolinsky, B., Juttner, R., Lehmann, T.N., Fahling, M., Schwarz, G., Legendre, P., and Meier, J.C. (2009). Splice-specific roles of glycine receptor alpha3 in the hippocampus. Eur J Neurosci 30, 1077-1091.

8.

Lynch, J.W., and Callister, R.J. (2006). Glycine receptors: a new therapeutic target in pain pathways. Curr Opin Investig Drugs 7, 48-53.

9.

Zeilhofer, H.U. (2005). The glycinergic control of spinal pain processing. Cell Mol Life Sci 62, 2027-2035.

10.

Zeilhofer, H.U., and Zeilhofer, U.B. (2008). Spinal dis-inhibition in inflammatory pain. Neurosci Lett 437, 170-174.

11.

Quinlan, J.J., Ferguson, C., Jester, K., Firestone, L.L., and Homanics, G.E. (2002). Mice with glycine receptor subunit mutations are both sensitive and resistant to volatile anesthetics. Anesth Analg 95, 578-582, table of contents.

12.

Ye, Q., Koltchine, V.V., Mihic, S.J., Mascia, M.P., Wick, M.J., Finn, S.E., Harrison, N.L., and Harris, R.A. (1998). Enhancement of glycine receptor function by ethanol is



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inversely correlated with molecular volume at position alpha267. J Biol Chem 273, 3314-3319. 13.

Yamakura, T., Bertaccini, E., Trudell, J.R., and Harris, R.A. (2001). Anesthetics and ion channels: molecular models and sites of action. Annu Rev Pharmacol Toxicol 41, 2351.

14.

Becker, K., Breitinger, H.G., Humeny, A., Meinck, H.M., Dietz, B., Aksu, F., and Becker, C.M. (2008). The novel hyperekplexia allele GLRA1(S267N) affects the ethanol site of the glycine receptor. Eur J Hum Genet 16, 223-228.

15.

Du, J., Lu, W., Wu, S., Cheng, Y., and Gouaux, E. (2015). Glycine receptor mechanism elucidated by electron cryo-microscopy. Nature 526, 224-229.

16.

Huang, X., Chen, H., Michelsen, K., Schneider, S., and Shaffer, P.L. (2015). Crystal structure of human glycine receptor-alpha3 bound to antagonist strychnine. Nature 526, 277-280.

17.

Breitinger, U., Raafat, K.M., and Breitinger, H.G. (2015). Glucose is a positive modulator for the activation of human recombinant glycine receptors. J Neurochem.

18.

Fucile, S., de Saint Jan, D., David-Watine, B., Korn, H., and Bregestovski, P. (1999). Comparison of glycine and GABA actions on the zebrafish homomeric glycine receptor. J Physiol 517 ( Pt 2), 369-383.

19.

De Saint Jan, D., David-Watine, B., Korn, H., and Bregestovski, P. (2001). Activation of human alpha1 and alpha2 homomeric glycine receptors by taurine and GABA. J Physiol 535, 741-755.

20.

Breitinger, U., and Breitinger, H.G. (2016). Augmentation of glycine receptor alpha3 currents suggests a mechanism for glucose-mediated analgesia. Neurosci Lett 612, 110115.

21.

Ulrich, P., and Cerami, A. (2001). Protein glycation, diabetes, and aging. Recent Prog Horm Res 56, 1-21.

22.

Asano, T., Katagiri, H., Tsukuda, K., Lin, J.L., Ishihara, H., Inukai, K., Yazaki, Y., and Oka, Y. (1992). Glucose binding enhances the papain susceptibility of the intracellular loop of the GLUT1 glucose transporter. FEBS Lett 298, 129-132.

23.

Higgins, P.J., and Bunn, H.F. (1981). Kinetic analysis of the nonenzymatic glycosylation of hemoglobin. J Biol Chem 256, 5204-5208. 20 ACS Paragon Plus Environment

Page 20 of 35

Page 21 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24.

Syrovy, I. (1994). Glycation of albumin: reaction with glucose, fructose, galactose, ribose or glyceraldehyde measured using four methods. J Biochem Biophys Methods 28, 115-121.

25.

Ledesma-Osuna, A.I., Ramos-Clamont, G., and Vazquez-Moreno, L. (2008). Characterization of bovine serum albumin glycated with glucose, galactose and lactose. Acta Biochim Pol 55, 491-497.

26.

Scheepers, A., Joost, H.G., and Schurmann, A. (2004). The glucose transporter families SGLT and GLUT: molecular basis of normal and aberrant function. JPEN J Parenter Enteral Nutr 28, 364-371.

27.

Li, Q., Manolescu, A., Ritzel, M., Yao, S., Slugoski, M., Young, J.D., Chen, X.Z., and Cheeseman, C.I. (2004). Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine. Am J Physiol Gastrointest Liver Physiol 287, G236-242.

28.

McPherson, J.D., Shilton, B.H., and Walton, D.J. (1988). Role of fructose in glycation and cross-linking of proteins. Biochemistry 27, 1901-1907.

29.

Gadgil, H.S., Bondarenko, P.V., Pipes, G., Rehder, D., McAuley, A., Perico, N., Dillon, T., Ricci, M., and Treuheit, M. (2007). The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations. J Pharm Sci 96, 2607-2621.

30.

Leslie, R.D., and Cohen, R.M. (2009). Biologic variability in plasma glucose, hemoglobin A1c, and advanced glycation end products associated with diabetes complications. J Diabetes Sci Technol 3, 635-643.

31.

Davison, A.S., Green, B.N., and Roberts, N.B. (2008). Fetal hemoglobin: assessment of glycation and acetylation status by electrospray ionization mass spectrometry. Clin Chem Lab Med 46, 1230-1238.

32.

MacDonald, J.A., Degenhardt, T., Baynes, J.W., and Storey, K.B. (2009). Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: in vivo and in vitro studies. Cryobiology 59, 223-225.

33.

Sen, S., Kar, M., Roy, A., and Chakraborti, A.S. (2005). Effect of nonenzymatic glycation on functional and structural properties of hemoglobin. Biophys Chem 113, 289-298.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

34.

Ladyzynski, P., Wojcicki, J.M., Bak, M.I., Sabalinska, S., Kawiak, J., Foltynski, P., Krzymien, J., and Karnafel, W. (2011). Hemoglobin glycation rate constant in nondiabetic Individuals. Ann Biomed Eng 39, 2721-2734.

35.

Absalom, N.L., Lewis, T.M., Kaplan, W., Pierce, K.D., and Schofield, P.R. (2003). Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor. J Biol Chem 278, 50151-50157.

36.

Shan, Q., and Lynch, J.W. (2012). Incompatibility between a pair of residues from the pre-M1 linker and Cys-loop blocks surface expression of the glycine receptor. J Biol Chem 287, 7535-7542.

37.

Johansen, M.B., Kiemer, L., and Brunak, S. (2006). Analysis and prediction of mammalian protein glycation. Glycobiology 16, 844-853.

38.

Hobbs, J.K., Jiao, W., Easter, A.D., Parker, E.J., Schipper, L.A., and Arcus, V.L. (2013). Change in heat capacity for enzyme catalysis determines temperature dependence of enzyme catalyzed rates. ACS Chem Biol 8, 2388-2393.

39.

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612.

40.

Sayle, R.A., and Milner-White, E.J. (1995). RASMOL: biomolecular graphics for all. Trends Biochem Sci 20, 374.


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Figure legends

Figure 1 Whole-cell current responses of α1glycine receptor expressed in HEK293 cells. Culture medium contained 5.5 mM of glucose. Glycine receptor treatment with glucose, fructose, galactose, sucrose and lactose: transfected cells were preincubated with sugars for 16 hours, electrophysiological measurements were performed in the absence of sugar. Treatment with sorbitol: measurements were done in external buffer containing 50 mM sorbitol. No sorbitol was present during expression. For more details see experimental section. Whole cell recordings were taken at 21-23 °C at -50 mV.

Figure 2 Concentration-response analysis of the modulation of α1 glycine receptors by saccharides. (A-F) Saccharides were added to the culture medium of HEK293 cells expressing homomeric α1 glycine receptors for the indicated time. Patch-clamp recordings were performed without sugar in the extracellular buffer. Open squares, solid line: 5.5 mM Glc (control = no sugar treatment); solid circles, dashed line: 2 mM additional sugar; solid up triangles, short dashed line: 10 mM additional sugar; solid down triangles, dash dotted line: 20 mM additional sugar; solid diamond, dash-dot-dotted line: 50 mM additional sugar. (A) glucose, (B) fructose, (C) mannose, (D) sucrose, (E) lactose, (F) galactose. (G) Modulation of recombinant glycine receptors by sorbitol. Open squares, solid line: 5.5 mM glucose (control = no sugar treatment); solid squares, dashed line: 50 mM sorbitol in the extracellular buffer during measurement; solid diamond, dash dotted line: 50 mM sorbitol were added to receptorexpressing HEK293 cells for 16 hours, followed by patch-clamp measurements in sugar-free extracellular buffer. See Table 2 for values of EC50 and Imax.



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Figure 3 Summary of modulation of glycine receptors by saccharides. (A) GlyR modulation by saccharides in the recording buffer. 50 mM glucose, fructose and sorbitol were added to the extracellular buffer during electrophysiological measurements. (BH) 1-50 mM of saccharide were added to the cell culture medium for 1 - 16 h during receptor expression, followed by patch-clamp measurements in the absence of sugar. Time of exposue to saccharide is indicated. (B) sorbitol, (C) glucose (D) fructose, (E) mannose, (F) galactose (G) sucrose and (H) lactose.

Figure 4 Kinetic analysis of glycine receptor modulation by saccharides. (A) Determination of time constants at different concentrations of saccharides. Solid squares, solid line: 50 mM; open diamond, dashed line: 20 mM; open down triangle, dash dotted line: 10 mM; open up triangle, dash dot dotted line: 2 mM; open circle, short dashed line: 1 mM. Saccharides are indicated. See text for constants. (B) Determination of the EC50 values of saccharides for GlyR modulation for glucose, fructose, mannose, sucrose and lactose. Solid symbols, solid line: 16 hrs pretreatment (50 mM); open symbols, dashed line: 1 h pretreatment (50 mM). Squares: 50 mM glucose; circles: 50 mM fructose; up triangles: 50 mM mannose; down triangles: 50 mM sucrose; diamonds: 50 mM lactose. (C) Left panel: Time constants for GlyR modulation by 50 mM of saccharide. Middle panel: EC50 values of saccharides for GlyR modulation. Right panel: comparison of end EC50 values of glycine after modulation by the indicated saccharides. All data were determined from kinetic analysis of the time dependence of GlyR modulation at 50 mM of saccharide. See Experimental Section for detail and Table 3 for constants. (D) Off kinetics of glycine receptor modulation by saccharides. Cells were treated with 50 mM glucose or 50 mM sucrose one day after transfection for 16 h. Additional saccharide was removed and cells kept in control medium 24 ACS Paragon Plus Environment

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(5.5 mM glucose) for another 24 h, followed by determination of glycine EC50 (see Methods). The glycine EC50 value after 16 h pretreatment with glucose was 12.8 ± 1.2 µM, increasing to 17.8 ± 1.1 µM after 24 h in control medium. Pretreatment with 50 mM sucrose gave glycine EC50 value of 16.8 ± 1.7 µM which increased to 21.7 ± 2.3 µM after 24 h in control medium. Significance of changes is indicated: * p < 0.05, ns: not significant (p > 0.05).

Figure 5 The role of residue α1(K143A) in GlyR modulation by glucose. (A) Glycine-evoked currents of the glycine receptor mutant α1(K143A) before (upper panel) and after (lower panel) pretreatment with 50 mM glucose. (B) Concentration-response curves of α1(K143A) receptors. Solid circles, solid line: 0 mM Glc; open circles, dashed line: pretreatment with 50 mM glucose for 16 h. (C) Summary of concentration-response data after glucose treatment. Mutant α1(K143A) channels were not sensitive to glucose modulation. (D) Structure of ivermectin-bound (left panel) and glucose-bound (right panel) glycine receptor. Subunits of GlyR are shown in backbone presentation and in different colors. The ligands and residue K143 are shown in stick presentation and colored according to the atom type.



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Table Legends Table 1 Modulation of GlyR responses by bath application of saccharides. HEK 293 cells were grown and transfected with GlyR α1 cDNA in control medium (1 g/l = 5.5 mM glucose), saccharides in the indicated concentration were added to the bath and perfusion buffer in patch-clamp experiments, thus cells were exposed to sugar for ~ 10 – 90 min prior to whole-cell recording.

*Data from (17)

Table 2 Modulation of GlyR responses by saccharides in the culture medium. Saccharides in the indicated concentration were added to the culture medium prior to patchclamp experiments. Incubation times and saccharide concentrations are indicated. Medium contained 1 g/l (5.5 mM) of glucose. For glucose, final concentrations are given, for other saccharides, indicated concentration plus 5.5 mM glucose were present. *Means ± SEM

**Data from (17)

***Fitted values of time-dependence of GlyR modulation at 50 mM of saccharide

Table 3 On-kinetics for the augmentation of GlyR currents by mono- and disaccharides. Constants are from a non-linear fit to a monoexponential decay of the time dependence of glycine EC50 values at 50 mM of saccharide (see Methods for detail).


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Tables Table 1 Modulation of GlyR responses by bath application of saccharides.

Sugar

EC50

Imax

(µ µM)

nA

0

39.3 ± 2.7

Glucose

50*

Fructose Sorbitol

Control – no sugar

Conc (mM)

n (cells)

Lit

2.0 ± 0.4

11

This study

15.5 ± 2.6

2.6 ± 0.6

6

(17)

50

12.8 ± 2.0

1.8 ± 0.9

4

This study

50

28.4 ± 6.8

2.3 ± 6.8

6

This study



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Table 2 Modulation of GlyR responses by saccharides in the culture medium. Saccharide

Conc (mM)

Control Monosaccharides Glucose

5.5 (TCS 5.5)

Fructose

Galactose Mannose

Sugar alcohol Sorbitol

Disaccharides Sucrose

Lactose

GlyR Mutant K143A (glucose)

Time (h)

EC50(Gly)* (µ µM)

Imax* nA

n (cells)

39.3 ± 2.7

2.0 ± 0.4

11

4.5 ± 0.9 1.4 ± 0.6 2.6 ± 0.6 1.7 ± 0.7 3.4 ± 0.5 1.1 ± 0.2 4.2 ± 0.5 1.3 ± 0.7 1.8 ± 0.4

4 5 5 3 6 5 6 3 6 9 3 5 4 5 6 5 4 5 9 5 5 4 4 4 4 4 3 4 7

Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Kinetic analysis***

Method

6.5 (TCS 6.5) 7.5 (TCS 7.5) 7.5 (TCS 7.5) 10 (TCS 10) 10 (TCS 10) 20 (TCS 20) 20 (TCS 20) 50 (TCS 50) 50** (TCS 50) 50 (TCS 50) 1 (TCS 6.5) 1 (TCS 6.5) 2 (TCS 7.5) 10 (TCS 15.5) 10 (TCS 15.5) 20 (TCS 25.5) 50 (TCS 55.5) 50**(TCS55.5) 50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5) 1 (TCS 6.5) 2 (TCS 7.5) 2 (TCS 7.5) 10 (TCS 15.5) 10 (TCS 15.5) 50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5)

16 1 16 1 16 1 16 1 16 16 1 16 16 1 16 16 1 16 16 1 16 16 1 16 1 16 1 16 16

24.9 ± 1.7 27.3 ± 1.9 11.4 ± 1.4 15.7 ± 3.6 14.0 ± 1.0 14.5 ± 0.4 12.1 ± 1.3 13.1 ± 1.1 12.8 ± 1.2 12.8 ± 0.7 33.3 ± 1.6 24.3 ± 1.9 11.6 ± 1.9 16.0 ± 0.8 14.2 ± 0.4 11.9 ± 0.7 11.8 ± 1.4 8.6 ± 0.5 9.4 ± 0.9 37.3 ± 3.0 35.4 ± 3.2 22.0 ± 2.6 27.4 ± 3.6 12.7 ± 2.3 16.7 ± 1.3 16.9 ± 2.3 15.0 ± 1.6 12.5 ± 0.5 12.5 ± 0.6

50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5)

1 4 16

33.2 ± 3.0 34.8 ± 7.4 34.4 ± 3.7

1.2 ± 3.0 0.4 ± 0.1 1.9 ± 0.5

8 4 6

Concentration-response curves Concentration-response curves Concentration-response curves

1 (TCS 6.5) 2 (TCS 7.5) 10 (TCS 15.5) 50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5) 1 (TCS 6.5) 2 (TCS 7.5) 10 (TCS 15.5) 50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5) 50 (TCS 55.5)

16 16 16 1 4 16 16 16 16 16 1 4 16 16

24.5 ± 2.6 17.6 ± 1.5 18.1 ± 2.7 30.1 ± 4.2 15.2 ± 0.7 16.8 ± 1.7 15.4 ± 2.7 23.3 ± 1.4 18.6 ± 0.9 18.5 ± 0.7 29.3 ± 7.4 16.4 ± 4.6 16.1 ± 1.2 15.3 ± 2.7

1.7 ± 0.8 1.8 ± 0.2 1.2 ± 0.6 2.4 ± 1.3 3.2 ± 0.4 2.3 ± 0.6

4 3 5 6 4 6 16 4 3 5 5 4 6 15

Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Kinetic analysis*** Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Kinetic analysis***

5.5 (TCS 5.5) 50 (TCS 55.5)

1

79.6 ± 2.5 78.7 ± 3.7

1.9 ± 0.5 5.1 ± 0.8

4 4

Concentration-response curves Concentration-response curves

3.8 ± 2.1 3.4 ± 1.5 2.8 ± 0.9 1.4 ± 0.3 2.1 ± 0.4 3.2 ± 0.9 0.7 ± 0.1 2.6 ± 0.2 2.9 ± 0.4 2.0 ± 1.0 4.2 ± 1.5 2.9 ± 0.6 2.3 ± 0.7 3.3 ± 0.8 2.9 ± 0.4 3.1 ± 1.0 2.3 ± 0.5

2.3 ± 0.4 3.4 ± 0.4 0.6 ± 0.1 1.7 ± 1.0 2.5 ± 0.4 1.3 ± 0.4


Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Kinetic analysis*** Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Concentration-response curves Kinetic analysis***

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Table 3 On-kinetics for the augmentation of GlyR currents by mono- and disaccharides. Sugar

Time constant h

EC50 (sugar) mM

End EC50 (Gly) µM

Monosaccharide Glucose Fructose Mannose

0.33 ± 0.17 0.44 ± 0.09 0.42 ± 0.07

0.88 ± 0.09 0.85 ± 0.24 0.78 ± 0.07

12.8 ± 0.7 9.4 ± 0.9 12.5 ± 0.6

Disaccharide Sucrose Lactose

1.63 ± 0.73 1.64 ± 0.37

0.69 ± 0.09 0.63 ± 0.17

15.4 ± 2.7 15.3 ± 2.7



Figure 1 Control G20

G50

G100 G1000

1 nA

G10

10 sec

Glucose G10

G20

G50

G10

G1000

G20

G50 G100 G1000

1 nA

1 nA

G5

Lactose

10 sec

10 sec

Fructose

Sucrose G50

G20

G1000

G10 G20

G50

G100

G1000

1 nA

1nA

G10

10 sec

10 sec

Sorbitol

Mannose G10

G50

G100

G1000

G10

G20

G50

G100

G1000

1 nA

G20

1 nA

10 sec

10 sec

Galactose G10

G20

G50

G1000

1 nA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10 sec


Page 30 of 35

Page 31 of 35

Figure 2 (A)

1.0

Glc Glc1h1h

Glc Glc16h

(F)

1,0

I / Imax

I / Imax

0.6 0.4

1

10

100

1

1000

10

100

1000

1

Gly (µM)

Gly (µM)

Frc Frc1h1h

Frc Frc16h

(G) I / Imax

I / Imax

0,4

1

10

100

1000

1

10

Gly (µM)

I / Imax

Man Man1h

1,0

0,8

0,8

0,6

0,6

0,4

0,4

0,2

0,2

0,4

100

1

1000

10

100

1000

1

10

100

1000

Gly (µM)

Suc 1h Suc

1,0 Suc Suc 4h

1,0 Suc Suc16h 16h

0,8

0,8

0,8

0,6

0,6

0,6

0,4

0,4

0,4

0,2

0,2

0,2 0,0

0,0

0,0 1

10

100

1

1000

10

100

1

1000

Gly (µM)

Gly (µM)

Lac 1h Lac

1,0

Lac Lac 4h

0,8

0,6

0,6

0,6

0,4

0,4

0,4

0,2

0,2

0,2

0,0

0,0 100

Gly (µM)

1000

100

1000

1,0 Lac Lac 16h 16h

0,8

10

10

Gly (µM)

0,8

1

0,0 1

10

100

10

100

Gly (µM)

Man Man16h

Gly (µM)

I / Imax

0,6

0,0 1

1,0

Sor Sor50 50mM

Gly (µM)

0,0

(E)

1,0

0,0

0,0

1,0

1000

0,2

0,2

(D)

100

0,8

0,6

1,0

10

Gly (µM)

0,8

(C)

0,4

0,0

0.0

1,0

0,6

0,2

0.2

(B)

Gal Gal50mM 50

0,8

0.8

I / Imax

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1000

1

10

100

1000

Gly (µM)

Gly (µM)


1000


Figure 3

n.s.

n.s.

40

EC50 Gly (µM)

EC50 Gly (µM)

40 30 20

**

**

10 0

C

50 mM Sorbitol 50mM Sorbitol

30 20 10 0

no Glc Frc Sor

Glucose – 16h

Glucose - 16h 40

40

n.s.

n.s.

Glucose – 1h

Glucose - 1h *

30 20

**

*

20

**

0

ctr 7.5 10 20 50

EC50 Gly (µM)

40

40

n.s.

30

30

20

**

** **

10

0

0

1

10

20

50

*

20

10 ctr

**

ctr 1

Frc (mM)

G 40

20

**

2 10 20 50

40

30 **

20

10

0

0

ctr

0

10

50

**

ctr

1

H

Lactose – 16h

**

**

20

0

1h 4h 16h Time

10

40

20 10 0

50

n.s.

30

** ** ** **

10 ctr

**

50 mM Lactose 50mM Lactose

Lactose - 16h

40 30

2

**

**

20

**

10

ctr 1 2 10 50 Lactose (mM)


0

ctr

1h

n.s. n.s.

30

Man (mM)

Man (mM)

10

ctr 1 2 10 50 Sucrose (mM)

2

**

20

10

n.s.

20

**

50 mMGalactose Galactose 50mM 40

40

*

30

F

Mannose – 16h Mannose - 16h

Mannose - 1h

30

** ** **

10 0

**

50 mMSucrose Sucrose 50mM

40

*

**

**

Glc (mM)

Mannose – 1h

Frc (mM)

Sucrose -–16h 16h Sucrose

30

E

Fructose – 16h

Fructose - 16h

EC50 Gly (µM)

Fructose – 1h

Fructose - 1h

**

ctr 6.5 7.5 10 20 50

Glc (mM)

D

**

** 10

10 0

ctr 1h 4h 16h

30 **

EC50 Gly (µM)

B

Bath Bath

EC50 Gly (µM)

A

EC50 Gly (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

4h 16h Time

ctr 1h 16h

Page 33 of 35

Figure 4 Fructose Fructose 40

30

30 20

20

10

10 0

5

10

0

15

40 30 20

55

10 10

time (hrs)

00

40

40

30

30

30

20

20

10

10

0

5

10

15

0

5

10

10

15

0

time (hrs)

time (hrs)

30 20

55

10 10

time (hrs)

0

15 15

5

10

15

time (hrs)

Lactose Lactose

40

20

40

10

15 15

Sucrose Sucrose

Sorbitol Sorbitol

Galactose Galactose

10 0 0

time (hrs)

EC50 Gly (µM)

Mannose Mannose

EC50 Gly (µM)

Glucose 40

EC50 Gly (µM)

EC50 Gly (µM)

A

5

10

15

time (hrs)

B 40

20

30

20

0.01

0.1

1

10

0.01

100

30

20

10

10

10

EC50 Gly (µM)

30

40

40

EC50 Gly (µM)

EC50 Gly (µM)

EC50 Gly (µM)

40

0.1

1

10

100

30

20

10 0.01

0.1

1

10

100

0.01

Mannose (mM)

Fructose (mM)

Glucose (mM)

0.1

1

10

**

1,5 1,0 n.s. n.s.

0,5 0,0

n.s. n.s. n.s.

1,0

n.s.

0,5

0,0

Glc Frc Man Lac Suc

Glc Frc Man Suc Lac

50 mM sugar

D

30

Sucrose

*

40

*

Sucrose * 40

n.s

20

10

10 on 24 h off

n.s

30

20

0 Ctr

n.s. n.s.

20

n.s. n.s. 10

0

Glc

Frc

Man

Suc

Lac

50 mM sugar - 16 hrs

Glucose

*

End Gly EC50

EC50 Sugar (mM)

Time constants (hrs)

**

2,0

0 Ctr

on 24 h off


100

Disaccharides (mM)

C

EC50 Gly (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Figure 5

A

B

K143A G20

G50

G100

G200 G1000

I / Imax

1 nA

no Glc

10 sec

G20

G50

G100

G200

G1000

1.0 α1(K143A) 0.8 0.6 0.4 0.2

50mM Glc

1 nA

0.0

C 50

10

α1 wildtype

100

40

80

30

60

20

**

10 0

α1(K143A)

40 20

5,5

100

Gly (µM)

10sec

n.s.

EC50 Gly (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

50

0

5,5

50

Glucose (mM)

D


1000

Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CN-2016-00044k For Table of contents use only Modulation of recombinant human α1 glycine receptors by mono- and disaccharides – a kinetic study Ulrike Breitinger, Heinrich Sticht and Hans-Georg Breitinger

ACS Paragon Plus Environment

Modulation of Recombinant Human Î±1 Glycine Receptors by Mono

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