Identification of residues critical for Cu2+-mediated inhibition of glycine α1 receptors

Zhenglan Chen, Glenn H. Dillon, Ren-Qi Huang

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7 Citations (Scopus)

Abstract

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 α1 receptors, with an IC50 of 4.1 ± 0.7 μM. 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.

Original languageEnglish
Pages (from-to)701-708
Number of pages8
JournalNeuropharmacology
Volume51
Issue number4
DOIs
StatePublished - 1 Sep 2006

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Glycine Receptors
Mutation
Histidine
Glycine
Divalent Cations
Threonine
Bromides
Alanine
Inhibitory Concentration 50

Keywords

  • Cu modulation
  • Glycine receptor
  • Histidine
  • MTSET
  • Site-direct mutagenesis
  • Zinc

Cite this

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title = "Identification of residues critical for Cu2+-mediated inhibition of glycine α1 receptors",
abstract = "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 α1 receptors, with an IC50 of 4.1 ± 0.7 μM. 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.",
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Identification of residues critical for Cu2+-mediated inhibition of glycine α1 receptors. / Chen, Zhenglan; Dillon, Glenn H.; Huang, Ren-Qi.

In: Neuropharmacology, Vol. 51, No. 4, 01.09.2006, p. 701-708.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Identification of residues critical for Cu2+-mediated inhibition of glycine α1 receptors

AU - Chen, Zhenglan

AU - Dillon, Glenn H.

AU - Huang, Ren-Qi

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N2 - 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 α1 receptors, with an IC50 of 4.1 ± 0.7 μM. 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.

AB - 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 α1 receptors, with an IC50 of 4.1 ± 0.7 μM. 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.

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KW - Histidine

KW - MTSET

KW - Site-direct mutagenesis

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DO - 10.1016/j.neuropharm.2006.05.009

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