Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

Abstract

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein ␤␥ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR ␣ 1 subunit that are critical for binding and functional modulation by G␤␥. Mutations within these sequences demonstrated that all of the residues detected are important for G␤␥ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are ␣-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein ␤␥ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.The ionotropic glycine receptors (GlyRs) 2 are members of the ligand-gated ion receptor superfamily, which includes inhibitory ␥-aminobutyric acid type A receptors and excitatory nAChR and 5-HT 3 receptors. These homologous receptors mediate fast synaptic transmission in the central nervous system (1, 2). Inhibitory GlyRs are critical for the control of excitability in the mammalian spinal cord and brain stem. Binding of glycine to the extracellular region induces a rapid increase in Cl Ϫ ion conductance, generating a hyperpolarization of the cell membrane (3-5). In neurons, the inhibitory action of GlyRs regulate several important physiological functions, like pain transmission, respiratory rhythms, motor coordination, and development (3-7). Like all members of the LGIC superfamily, GlyRs are pentamers composed of five subunits in which each subunit possesses four transmembrane domains arranged to form the ion pore. In this structure, the individual subunits provide extracellular and intracellular domains that play roles in ligand binding and intracellular modulation, respectively (1-3, 5).The function of GlyRs can be effectively modulated by extracellularly acting compounds like strychnine, picrotoxin, zinc ions, and ethanol (3-5, 8, 9). Furthermore, the receptor can also be modulated by intracellular signaling. One of the most studied and recognized pathways involved in regulation of ligandgated ion channel function are phosphorylation processes through protein kinases. Indeed, GlyR and other members of the LGIC superfamily are modulated by activation of cAMPdependent kinases and protein kinase C (10). This involves specific serine residues in the loop between transmembrane domains 3 and 4 (6, 10 -14). On the other hand, recent reports have shown that the activity of GlyRs and nAChRs can be modulated by G protein ␤␥ subunits in a phosphorylation-independent manner (15, 16). In both cases, activation of G proteins, using nonhydrolyzable GTP analogs or by application of purified G␤␥ dimers, generates a strong e...

Functional Complementation ofGlra1spd-ot, a Glycine Receptor Subunit Mutant, by Independently Expressed C-Terminal Domains

“…1C). The corresponding wild-type sequence contains a basic motif involved in surface integration and G-protein ␤␥ binding (Sadtler et al, 2003;Yevenes et al, 2006). As evident from human hyperekplexia mutations, the cytosolic arginine residues flanking transmembrane regions 2 and 4 are important for GlyR biogenensis and channel function (Langosch et al, 1993;Rea et al, 2002).…”

“…Members of this superfamily share an Ig-like fold of the N terminus and four transmembrane domains (TMs), with TM2 lining the ion pore. Within the intracellular TM3-TM4 loop, a basic motif was identified mediating accumulation of receptors at the cell surface (Sadtler et al, 2003;Yevenes et al, 2006).…”

Blockade of Ethanol-Induced Potentiation of Glycine Receptors by a Peptide That Interferes with Gβγ Binding

“…The IL is relevant for GlyR function because it was reported that it can be modulated by activation of intracellular cAMP-dependent protein kinase and protein kinase C (Aguayo et al, 1996;Tapia et al, 1997). GlyR modulation and the direct interaction with specific intracellular domains in the receptor have been recently documented (Yevenes et al, 2003(Yevenes et al, , 2006. Furthermore, recent studies showed that, similar to nicotinic acetylcholine receptor (Fischer et al, 2005), the GlyR function can be directly modulated by G␤␥.…”

“…In the case of GlyRs, mutations in basic amino acids in the IL decreased both the interaction and modulation of G␤␥ with GlyRs (Yevenes et al, 2006). Therefore, using biochemical, electrophysiological, and in silico techniques, we decided to examine the ability of several regions of the ␣ 1 GlyR IL to directly interfere with G␤␥ signaling.…”

Multifunctional Basic Motif in the Glycine Receptor Intracellular Domain Induces Subunit-specific Sorting

“…By interaction with the dynein complex, gephyrin finally mediates vesicular retrograde transport of the receptor along microtubules (12). For the TM3-4 loops of GlyR␣ subunits, a number of protein-protein interactions that are guided by consensus motifs have been described (13,14). However, the roles of these interactions and consensus motifs in trafficking of the receptor remain elusive.…”