Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

Timothy Lynagh; Timothy I. Webb; Christine L. Dixon; Brett A. Cromers; Joseph W. Lynch

doi:10.1074/jbc.m111.262634

Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

Timothy Lynagh, Timothy I. Webb, Christine L. Dixon, Brett A. Cromers, Joseph W. Lynch^*

^*Corresponding author for this work

42 Citations (Scopus)

Abstract

Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala ²⁸⁸ and Pro ²³⁰ as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu ²¹⁸, Ser ²⁶⁰, and Thr ²⁸⁵ (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser ²⁶⁰ and Thr ²⁸⁵ are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.

Original language	English
Journal	Journal of Biological Chemistry
Volume	286
Issue number	51
Pages (from-to)	43913-43924
Number of pages	12
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.m111.262634
Publication status	Published - 23 Dec 2011

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title = "Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel",

abstract = "Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala 288 and Pro 230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu 218, Ser 260, and Thr 285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser 260 and Thr 285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.",

author = "Timothy Lynagh and Webb, {Timothy I.} and Dixon, {Christine L.} and Cromers, {Brett A.} and Lynch, {Joseph W.}",

year = "2011",

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doi = "10.1074/jbc.m111.262634",

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T1 - Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

AU - Lynagh, Timothy

AU - Webb, Timothy I.

AU - Dixon, Christine L.

AU - Cromers, Brett A.

AU - Lynch, Joseph W.

PY - 2011/12/23

Y1 - 2011/12/23

N2 - Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala 288 and Pro 230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu 218, Ser 260, and Thr 285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser 260 and Thr 285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.

AB - Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala 288 and Pro 230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu 218, Ser 260, and Thr 285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser 260 and Thr 285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.

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U2 - 10.1074/jbc.m111.262634

DO - 10.1074/jbc.m111.262634

M3 - Journal article

C2 - 22033924

AN - SCOPUS:83755162397

SN - 0021-9258

VL - 286

SP - 43913

EP - 43924

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 51

ER -

Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

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