Agonist-induced closure of constitutively open γ-aminobutyric acid channels with mutated M2 domains

Abstract

Ligand-gated ion channels display a fundamental property-channels remain virtually closed at rest and open upon agonist binding. Here we show that substituting alanines for either of two amino acid residues (T314 or L317) in the M2 region of the ␥-aminobutyric acid (GABA) 1 subunit results in spontaneous channel opening in the absence of ligand. Surprisingly, for two single point mutants (T314A or L317A), application of very low concentrations of agonist partially suppressed this spontaneous current, while higher concentrations re-activated the receptors. When both of these sites were mutated (T314A͞L317A), GABA nearly completely suppressed the constitutive current and did not re-activate the current even at very high concentrations. This study provides important new insights into the structure-function relationship of ligand-gated ion channels, where modification of the structure of the channel pore region not only alters the allosteric transition of the receptor protein but also reverses the polarity of agonist regulation of channel gating. Our results suggest that the sites where these two residues are located are structurally critical for channel gating.Members of the ligand-gated ion channel superfamily share certain structural and functional similarities (1-5) and include the following receptors: nicotinic acetylcholine (nACh), glycine, 5-hydroxytryptamine type 3, and ␥-aminobutyric acid (GABA, A and C subtypes). In this family, the receptor is apparently composed of five subunits, and the second membrane-spanning segment (M2) forms the pore of the channel (6-8). Typically, the channel is closed at rest and opens upon agonist binding to its receptor site, located at some distance from the pore region. Channel activity is thought to be controlled by a gate inside the pore (7-9). The structure of the nACh receptor (nAChR) channel in the closed and open states has been characterized at 9-Å resolution in electron crystallographic studies (9, 10). Receptor protein function has been interpreted in terms of an allosteric transition model (11,12). However, the nature of the channel gate as well as the exact mechanism of ligand regulation of gating are still not clear.The GABA subunits are known to comprise, at least in part, the recently described GABA C receptor (5, 13-17). A threonine residue lies at position 314 in the M2 domain of the 1 subunit and is conserved in most known GABA and glycine receptor subunits (Fig. 1). Previously, we reported that a naturally occurring mutation (a methionine instead of a threonine) at the corresponding site of the 2 subunit is responsible for the resistance to picrotoxinin (PTX) blockade of native GABA C receptors in the rat retina (16). To explore further the role of this and surrounding sites on receptor-channel function of GABA C receptors, we made a series of additional substitutions by site-directed mutagenesis, taking advantage of the observation that recombinant 1 subunits are in general capable of forming functional homomeric receptors (5, 16). Here we report t...

An Engineered Glutamate-gated Chloride (GluCl) Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin

“…1A). Weak agonist-induced signals were the result of elevated base-line fluorescence (data not shown), suggesting an increased level of background conductance, which has been reported for some leucine 9Ј mutations in other Cysloop receptors (13,20,(22)(23)(24)(25). The L9ЈF mutant retained a maximal response comparable with that of WT-XFP.…”

Thr339-to-Serine Substitution in Rat P2X2Receptor Second Transmembrane Domain Causes Constitutive Opening and Indicates a Gating Role for Lys308

“…Thus, several ligand-gated channels have been reported to open spontaneously when (relatively minor) mutations have been introduced into their structure. Some examples for homomeric channels are GABA receptors (Pan et al, 1997;Torres and Weiss, 2002), glycine ␣1 receptors (Dupre et al, 2007), nicotinic ␣7 receptors (Bertrand et al, 1997), and glutamate ␦2 receptors (Zuo et al, 1997). The approach is of course subject to the criticism that the open state of the mutated channel is surely not identical with that of the liganded channel.…”

Allosteric activation mechanism of the cys-loop receptors

“…Studies using sitedirected mutagenesis and ultrastructural analysis have identified the second transmembrane domain (M2) as the pore-lining domain in the cys-loop receptors. Hydrophilic substitutions of the conserved leucine in the mid-point of the M2 domain dramatically influence channel gating kinetics, increase agonist sensitivity, and create spontaneous opening channels in several members of the cys-loop receptors [11][12][13][33][34][35][36][37][38][39] . Although earlier studies using cysteine accessibility test suggested that the gate is in the intracellular end of M2 [39,40] , it is now clear that the accessibility of these residues in the intracellular end of M2 in the absence of agonist is due to spontaneous opening of the M2 mutant channel [41] .…”