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  • br Conclusion br Experimental procedures br Introduction The

    2021-11-23


    Conclusion
    Experimental procedures
    Introduction The glycine receptor (GlyR) is a member of the Cys-loop family of ligand-gated ion channels. It is the primary inhibitory receptor in the brainstem and spinal cord but also plays important roles in higher igf 1 inhibitor regions, including the hippocampus, nucleus accumbens and prefrontal cortex (Baer et al., 2009, Jonsson et al., 2012, Jonsson et al., 2009, Lynch, 2004). GlyRs are pentameric in structure with the five subunits arranged around a central anion-conducting channel. Thus far, four alpha subunits and one beta subunit have been identified, of which α1–3 and β are found in humans. GlyRs can express either as homomeric receptors composed solely of α subunits, or as αβ heteromeric receptors with a stoichiometry of 2α:3β (Betz et al., 1993, Bowery and Smart, 2006, Lynch, 2004). In the spinal cord a developmental switch occurs from prenatal α2 homomers to α1β receptors in the adult. However, in the forebrain it appears that the α2 subunit continues to be expressed into adulthood, along with the β subunit (Jonsson et al., 2012). GlyR activity is affected by a large variety of allosteric modulators including zinc, alcohols, anesthetics and inhaled drugs of abuse (Beckstead et al., 2000, Harvey et al., 1999, McCracken et al., 2010, McCracken et al., 2013, Mihic et al., 1997), making it a promising clinical target for the treatment of alcohol and drug addiction (Söderpalm and Ericson, 2013, Tipps et al., 2010). Zinc is present endogenously at nanomolar concentrations known to enhance GlyR function. It exhibits biphasic actions at GlyRs, potentiating currents at concentrations <10μM, while higher concentrations produce inhibition (Harvey et al., 1999, Laube et al., 2000). Taurine is a partial agonist of the GlyR, with approximately 5% the efficacy of glycine (Lape et al., 2008), and is believed to be an important GlyR agonist in a number of brain regions (Albrecht and Schousboe, 2005). For example, Mori et al. (2002) showed that an uptake inhibitor of taurine induced a strychnine-sensitive chloride current in hippocampal organotypic slice cultures. Previous research has largely focused on allosteric modulation at glycine-activated receptors. Modulators shift glycine concentration–response curves either to the left or to the right but have no effects at maximally-effective glycine concentrations. However, Kirson et al., 2013, Kirson et al., 2012 showed that ethanol, volatile anesthetics, inhaled drugs of abuse and zinc are able to enhance currents elicited by maximally-effective concentrations of taurine, but not glycine. This suggested that these modulators had an effect on the probability of taurine-activated channel opening (Po), which would already be near maximum when a saturating concentration of glycine was tested. Most studies of allosteric modulation are performed using concentrations of agonists that are low on their concentration–response curves, since it is at these agonist concentrations that the greatest modulatory effects are seen. In this study we investigated whether agonist efficacy also affects the magnitude of zinc and ethanol enhancement of GlyR function seen under those conditions.
    Results Zinc was tested for its enhancing effects of α1 homomeric GlyR currents elicited by submaximal concentrations of glycine or taurine. Concentrations of each agonist were first identified that produced equal currents, corresponding to 5–10% of the maximally-effective glycine response (EC5–10 glycine). In order to do so the EC5–10 concentration of glycine was first identified in each oocyte and then the concentration of taurine producing a similar current was determined (Fig. 1A). Where that concentration of taurine fell on the taurine concentration–response curve was next determined, relative to a maximally-effective concentration of taurine (100mM). Concentrations of 84±4μM glycine had EC values of 6.28±0.70 relative to 10mM glycine, while concentrations of 1.2±0.2mM taurine, producing currents of the same magnitude in each oocyte as glycine, had EC values of 22.69±3.98 relative to 100mM taurine. The concentrations of taurine used thus fell significantly higher on their concentration–response curves than the concentrations of glycine did on theirs [t(4)=4.06, p<0.005] (Fig. 1B).