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    • We next compared the enhancing effects of nM zinc

    2021-11-06

    We next compared the enhancing effects of 100nM zinc on currents produced by a low concentration of glycine with zinc effects on the same currents produced by the partial agonist taurine. In this experiment, zinc was co-applied with concentrations of glycine or taurine producing 5–10% of the maximally effective glycine response; i.e., the absolute currents produced by glycine and taurine were similar (Fig. 2A). Zinc-potentiated currents were compared to those produced by agonist alone as shown in Fig. 2B. Co-application with zinc resulted in a significant enhancement of both glycine- and taurine-mediated currents [F(1,23)=24.12, p<0.001]. There was, however, no difference in the degree of zinc enhancement seen between glycine and taurine [F(1,23)=1.19, p>0.28]. When data from individual oocytes were plotted (Fig. 2C), there was a large degree of variation in zinc enhancement seen. Previous studies revealed that the Memantine hydrochloride used in our studies contain nanomolar levels of contaminating zinc, sufficient to affect GlyR function (Cornelison and Mihic, 2014). We hypothesized that the degree of variability within our data set might be attributable to variations in this amount of contaminating zinc between the different preparations of buffers used in this experiment. In order to minimize the effects that variable background levels of zinc might have on our results, we repeated our previous experiment using a much higher concentration of zinc (2.5μM) applied with a fixed (2.5mM) concentration of the zinc chelator, tricine. This resulted in much lower inter-experiment variability in zinc enhancement but, again, no differences were seen in zinc effects on glycine- vs. taurine-activated GlyR (Fig. 3A and B) [F(1,15)=0.62, p>0.44]. We next determined how the percent enhancement of taurine-activated GlyR currents by zinc varied with taurine and glycine concentration. The average zinc percent potentiation and taurine EC values were plotted on the ordinate against taurine concentration (Fig. 4A). A concentration of 2.5μM zinc (+2.5mM tricine) enhanced taurine responses in a manner that was dependent on the concentration of taurine used [F(5,38)=2.55, p<0.05], with less enhancement seen at higher taurine concentrations. It should be noted, however, that even at saturating taurine concentrations, zinc still had a potentiating effect. When glycine was the agonist (Fig. 4B), zinc again enhanced GlyR currents in a concentration-dependent manner [F(4,19)=16.29, p<0.001], but this time with negligible enhancement seen at higher glycine concentrations. Zinc produced no enhancement at EC50 glycine concentrations and above, in contrast to the clear enhancement still seen at the corresponding taurine concentrations. When zinc enhancement was plotted against each agonist׳s EC value (relative to its own maximal effect), greater zinc-mediated potentiation was seen at all concentrations of taurine (Fig. 4C). If only data obtained using low (less than EC20) concentrations of taurine and glycine are compared, even then significantly greater zinc enhancement is seen with taurine [t(28)=3.21, p<0.004]. In order to determine if the effects seen in our previous experiments were unique to zinc, we tested another allosteric modulator, ethanol, in place of zinc. Co-application of 200mM ethanol had a significant potentiating effect on glycine- and taurine-mediated currents [F(1,19)=123.22, p<0.001]. However, again there was no difference seen in the degree of potentiation between glycine and taurine [F(1,19)=0.001, p>0.97] (Fig. 5A), despite the significant difference [t(8)=3.2, p<0.014] in their respective agonist EC values (Fig. 5B, inset). All assays in this experiment were performed using a single preparation of MBS so that background contaminating levels of zinc would be constant throughout the experiment. Data shown for individual oocytes (Fig. 5B) show less variability than seen previously when the concentration of contaminating zinc was not controlled (Fig. 2C). A lower concentration (50mM) of ethanol was also tested using this same experimental paradigm, and similar results were found. Ethanol enhanced glycine-mediated currents by 15.6±2.2%, and taurine-mediated currents by 18.1±5%, when the two agonists produced currents of the same magnitude, and thus when the concentrations of taurine used fell significantly higher on their concentration–response curves than did the concentrations of glycine on theirs.