Molecular determinants of glycine receptor alphabeta subunit sensitivities to Zn2+-mediated inhibition.
Glycine receptors exhibit a biphasic sensitivity profile in response to Zn2+-mediated modulation, with low Zn2+ concentrations potentiating (< 10 microm), and higher Zn2+ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn2+-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) alpha1 subunit compared to either GlyR alpha2 or alpha3 subtypes. Swapping the divergent histidine (H107) residue in GlyR alpha1, which together with the conserved H109 forms part of an intersubunit Zn2+-binding site, for the equivalent asparagine residue present in GlyR alpha2 and alpha3, reversed this phenotype. Co-expression of heteromeric GlyR alpha1 or alpha2 with the ancillary beta subunit yielded receptors that maintained their distinctive sensitivities to Zn2+ inhibition. However, GlyR alpha2beta heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR alpha2 homomer. Comparative studies to elucidate the specific residue in the beta subunit responsible for this differential sensitivity revealed instead threonine 133 in the alpha1 subunit as a new vital component for Zn2+-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated beta subunit could indeed affect Zn2+-mediated inhibition but only from one side of the intersubunit Zn2+-binding site, equivalent to the GlyR alpha1 H107 face. This strongly suggests that the alpha subunit is responsible for Zn2+-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn2+-binding site where H109 and T133 are located.
Miller
Paul S
PS
Department of Pharmacology, University College London, Medical Sciences Building, Gower Street, London WC1E 6BT, UK.
Beato
Marco
M
Harvey
Robert J
RJ
Smart
Trevor G
TG
D001665:Binding Sites; D002460:Cell Line; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D021122:Protein Subunits; D018009:Receptors, Glycine / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology*
D021122:Protein Subunits; D018009:Receptors, Glycine; D015032:Zinc