Purification and characterization of the glycine receptor of pig spinal cord

Abstract

A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.

Baculovirus‐driven expression and purification of glycine receptor α1 homo‐oligomers

“…In a typical purification experiment, about 30% of the [3H]strychnine binding sites applied to the column were recovered in the eluate, and the final yield of the binding sites in the glycine eluate was about 10% of the sites present in unsolubil- ized membranes. This is comparable to the yield obtained with GlyR solubilized from mammalian spinal cord membranes [10,17]. In absolute numbers, one liter of cell culture containing about 1 × 109 cells, yielded about 400 pmol of affinity purified [3H]strychnine binding sites.…”

What confers specificity on glycine for its receptor site?

“…The responses induced by glycine, Tau, and other active a-and #-amino acids were preferentially inhibited by the specific glycine receptor antagonist, strychnine (Curtis et al, 1968;Graham et al, 1985). On the other hand, the selective GABAA receptor antagonist, bicuculline (Curtis et al, 1971;Akaike et al, 1985;Yakushiji et al, 1987), the Cl-channel blocker, picrotoxin (Constanti, 1978;Yakushiji et 1987) and the Tau receptor antagonist, TAG (Martin et al, 1981;Yarbrough et al, 1981;Okamoto et al, 1983) failed to antagonize the a-and fl-amino acidinduced Im.…”

“…In spite of its simple chemical structure, many kinds of glycine binding sites have been reported in vertebrate tissues: (a) a conventional glycine receptor which operates through a Cl-channel and which is sensitive to strychnine (Curtis et al, 1968;Graham et al, 1985), (b) a strychnine-insensitive glycine binding site (De Feudis, 1977;Bristow et al, 1986), one of which may allosterically regulate the Nmethyl-D-aspartate (NMDA) receptor (Johnson & Ascher, 1987), and (c) a glycine transport carrier (Johnson & Iversen, 1971;Wilkin et al, 1981). In general, for compounds with complex chemical structures and flexibility in the molecule, there exists the possibility of activity at different recognition sites, due to their conformational variation.…”

Isoform Heterogeneity of the Human Gephyrin Gene (GPHN), Binding Domains to the Glycine Receptor, and Mutation Analysis in Hyperekplexia

“…First identified as a 93-kDa protein by co-purification with GlyRs from rat spinal cord, GPHN demonstrated high affinity binding to tubulin (15)(16)(17). It is suggested that gephyrin forms a postsynaptic organizational lattice structure that dynamically immobilizes and clusters inhibitory receptors onto the cytoskeletal infrastructure (14,18).…”