A Novel Glycine Receptor β Subunit Splice Variant Predicts an Unorthodox Transmembrane Topology

Abstract

The inhibitory glycine receptor is a ligand-gated ion channel with a pentameric assembly from ligand binding ␣ and structural ␤ subunits. In addition to ␣ subunit gene variants (␣1-␣4) and developmental alterations in subunit composition of the receptor protein complex, alternative splicing of ␣ subunits has been found to contribute to glycine receptor heterogeneity. Here, we describe a novel splice variant of the glycine receptor ␤ subunit from mouse central nervous system, prevailing in macroglial cells, predominantly in astrocytes and extraneural tissues. As predicted by its cDNA sequence, the novel subunit ␤⌬7 lacks amino acid positions 251-302 encoded by exon 7 of the Glrb gene. Transcripts and antigen of ␤⌬7 were detected in cerebral cortex, liver, and heart. Lack of exon 7 results in a profoundly altered prediction of transmembrane topology as ␤⌬7 lacks TM1 and TM2 present in the full-length variant. Despite these topological alterations, in vitro studies showed that the ␤⌬7 polypeptide integrates into the plasma membrane, forming receptor complexes with the ␣1 subunit and gephyrin. Our data demonstrate that a topology deviating from the classical four transmembrane-fold is compatible with formation of glycine receptor protein complexes. However, co-expression of ␣1 with ␤⌬7 subunits did not change glycine receptor channel properties. Rather, the high level of expression in non-neuronal cells having intimate contact with synaptic regions may account for a yet unknown function of this splice variant ␤⌬7 in glycinergic neurotransmission. Glycine receptors (GlyRs)2 belong to the superfamily of Cysloop receptors, which also include nicotinic acetylcholine receptors, ionotropic ␥-aminobutyric acid receptors (GABA A and GABA C ), and the ionotropic serotonin receptor subtype 5HT 3 (1). GlyRs are pentameric assemblies of five subunits surrounding a central ion-conducting pore that mediates rapid inhibitory neurotransmission in the spinal cord and brainstem (2, 3). Common features of all Cys-loop receptor subunits include four transmembrane regions (TM1-TM4), where TM2 forms the ion channel pore, and a large extracellular N-terminal ligand-binding domain. This domain shows significant structural homology to the acetylcholine-binding protein of Lymnaea stagnalis (4), a member of the immunoglobulin-like superfamily of proteins. By association of ␤ subunits with the intracellular anchor protein gephyrin, GlyRs are clustered at the postsynaptic membrane. The gephyrin binding motif of the ␤ subunit polypeptide has been mapped to the long intracellular TM3-4 loop (5-9).The GlyR ␤ subunit gene is widely transcribed throughout the central nervous system of neonatal and adult rodents. Starting at embryonic day 14, ␤ transcripts are first detectable by in situ hybridization in rat spinal cord and telencephalon (10, 11). Expression of the ligand binding ␣ subunit variants (␣1-␣4) is highly regulated during development (12, 13). Further GlyR subunit heterogeneity is generated by alternative splicing and RNA editing (␣1 and ␣...

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

“…Finally, cells were embedded in Mowiol and subjected to confocal microscopy on a DMIRE2 confocal microscope (Leica, Wetzlar). Electrophysiology was carried out essentially as described (22). Under the conditions used, recombinant GlyRs showed little desensitization, consistent with earlier reports (23).…”

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

“…Assays were performed with transfected COS7 cells 48 h after transfection as described previously (Oertel et al, 2007).…”

Molecular architecture of glycinergic synapses

“…Splice errors in the GLRB gene can lead to compound heterozygote mutations, as found in patients with hyperekplexia (Oertel et al 2007;Rees et al 2002). Recently, a novel splice variant lacking exon 7 (encoding TM 1 and 2) of the transcript ( 7) has been described in glial and other non-neuronal cells (Oertel et al 2007). Over-expression of this polypeptide in heterologous cells, either alone or in combination with the 1 subunit, shows that it integrates into the cell membrane and associates with the 1 subunit and gephyrin.…”

“…Over-expression of this polypeptide in heterologous cells, either alone or in combination with the 1 subunit, shows that it integrates into the cell membrane and associates with the 1 subunit and gephyrin. These properties are interesting, given that this splice variant lacks TM1 and TM2 due to the splicing event, while its physiological properties subunits remain unchanged upon co-expression with the 1 subunit (Oertel et al 2007). Shown are two models, which diVer in the stoichiometric ratios of their and subunits.…”