An Amino Acid Substitution in the Pore Region of a Glutamate-gated Chloride Channel Enables the Coupling of Ligand Binding to Channel Gating

Adrian Etter,Doris F Cully,James M Schaeffer,Ken K Liu,Joseph P Arena

doi:10.1074/jbc.271.27.16035

Abstract

Many of the subunits of ligand-gated ion channels respond poorly, if at all, when expressed as homomeric channels in Xenopus oocytes. This lack of a ligand response has been thought to result from poor surface expression, poor assembly, or lack of an agonist binding domain. The Caenorhabditis elegans glutamate-gated chloride channel subunit GluClbeta responds to glutamate as a homomeric channel while the GluClalpha subunit is insensitive. A chimera between GluClalpha and GluClbeta was used to suggest that major determinants for glutamate binding are present on the GluClalpha N terminus. Amino acid substitutions in the presumed pore of GluClalpha conferred direct glutamate gating indicating that GluClalpha is deficient in coupling of ligand binding to channel gating. Heteromeric channels of GluClalpha+beta may differ from the prototypic muscle nicotinic acetylcholine receptor in that they have the potential to bind ligand to all of the subunits forming the channel.

Highlights

Ligand-gated ion channels form a gene superfamily composed of cation channels gated by acetylcholine and serotonin, and anion channels gated by ␥-aminobutyric acid (GABA),1 glycine, and glutamate [1,2,3,4,5,6]
The prototype of a ligand-gated ion channels is the muscle nicotinic acetylcholine receptor where the major determinants of ligand binding are located on the ␣ subunits [2,3,4]
A reduction in the free energy necessary to couple ligand binding to channel gating has been observed for point mutations in the second membrane spanning domain of nicotinic acetylcholine receptors [24, 25]

Summary

Introduction

Ligand-gated ion channels form a gene superfamily composed of cation channels gated by acetylcholine and serotonin, and anion channels gated by ␥-aminobutyric acid (GABA), glycine, and glutamate [1,2,3,4,5,6]. GluCl␣ and GluCl␤ are subunits of a glutamate-gated chloride channel (GluCl) from the nematode Caenorhabditis elegans [1]. They are 45% identical on the amino acid level, and closely related to the glycine and GABAA receptor families [1]. When co-expressed in oocytes, GluCl␣ and GluCl␤ form heteromeric channels gated by glutamate and the widely used antiparasitic agent ivermectin [1]. Homomeric GluCl␤ channels are directly gated with glutamate demonstrating that GluCl␤ contains all the determinants for ligand binding and coupling to channel gating [1]. Heteromeric channels of GluCl␣ϩ␤ have the potential to bind ligand to all of the subunits forming the channel

Methods

Results

Conclusion