Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors.
1. The effects of Zn2+ on glycine receptor (GlyR) currents were analysed in Xenopus oocytes and human embryonic kidney cells expressing homomeric human wild-type and mutant alpha1 subunit GlyRs. 2. Low concentrations (10 microM) of extracellular Zn2+ converted the partial agonist taurine into a high-efficacy agonist. Concentration-response analysis showed that the EC50 for taurine decreased whereas the Hill coefficient increased under these conditions. In contrast, 50-500 microM Zn2+ showed an increased EC50 value and reduced maximal inducible taurine currents. The potency of competitive antagonists was not affected in the presence of Zn2+. 3. Single-channel recording from outside-out patches revealed different kinetics of glycine- and taurine-gated currents. With both agonists, Zn2+ altered the open probability of the alpha1 GlyR without changing its unitary conductance. Low Zn2+ concentrations (5 microM) increased both the opening frequency and mean burst duration, whereas higher Zn2+ concentrations (> 50 microM) reduced GlyR open probability mainly by decreasing the open frequency and the relative contribution of the longest burst of the single-channel events. 4. Site-directed mutagenesis of the GlyR alpha1 subunit identified aspartate 80 and threonine 112 as important determinants of Zn2+ potentiation and inhibition, respectively, without affecting potentiation by ethanol. 5. Our data support the view that Zn2+ modulates different steps of the receptor binding and gating cycle via specific allosteric high- and low-affinity binding sites in the extracellular N-terminal region of the GlyR alpha1 subunit.
Laube
B
B
Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, 60528 Frankfurt am Main, Germany. lauge@mpih-frankfurt.mpg.de
Kuhse
J
J
Betz
H
H
D000465:Algorithms; D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018075:RNA, Complementary / Q000235:genetics; D018009:Receptors, Glycine / Q000819:agonists / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013654:Taurine / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis; D015032:Zinc / Q000494:pharmacology*
D018075:RNA, Complementary; D018009:Receptors, Glycine; D011994:Recombinant Proteins; D013654:Taurine; D015032:Zinc