Identification of residues critical for Cu2+-mediated inhibition of glycine alpha1 receptors.
Endogenous divalent cations Cu2+ and Zn2+ suppress the activity of glycine receptors (glyRs). Whereas residues critical for the effects of Zn2+ on glyRs have been identified, little is known about the determinants of Cu2+-mediated inhibition. In the present studies, we have assessed the potential commonality of Zn2+ and Cu2+-mediated inhibition of glyRs. Cu2+ potently inhibited recombinant human glycine alpha1 receptors, with an IC50 of 4.1+/-0.7 microM. Systematic mutation of extracellular histidine residues revealed that mutation H215A greatly reduced the inhibitory modulation by Cu2+. Substitution of H215 with C produced receptors with Cu2+ sensitivity similar to the wild type. Furthermore, modification of H215C with a thio-specific reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), reduced Cu2+ sensitivity of H215C receptors. However, mutation of other extracellular histidine residues including H107 and H109, which are known inhibitory Zn2+coordination sites, failed to influence inhibition of glycine currents by Cu2+. Moreover, mutation to alanine of two threonine residues (T112, T133) critical for Zn2+ inhibition had no effect (T133A) or only partial inhibitory effects (T112A) on Cu2+-induced inhibition. The double mutation, T112A/H215A, caused greater effects on Cu2+-mediated inhibition than either mutation alone. In addition, the glycine currents recorded from T112A/H215A mutant receptors were significantly potentiated by low concentrations of Cu2+. Our results have identified critical determinants of Cu2+-mediated inhibition of glyRs. Moreover, we demonstrate for the first time a clear difference in residues responsible for Cu2+-mediated compared to Zn2+-mediated inhibition of glyRs.
Chen
Zhenglan
Z
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
Dillon
Glenn H
GH
Huang
Renqi
R
D002461:Cell Line, Transformed; D003001:Cloning, Molecular / Q000379:methods; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005998:Glycine / Q000494:pharmacology; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008564:Membrane Potentials / Q000187:drug effects; D008698:Mesylates / Q000494:pharmacology; D016296:Mutagenesis / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D018009:Receptors, Glycine / Q000737:chemistry* / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D015032:Zinc / Q000494:pharmacology
D008698:Mesylates; D018009:Receptors, Glycine; D013439:Sulfhydryl Reagents; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D006639:Histidine; D003300:Copper; D015032:Zinc; D005998:Glycine