Werner Hoch

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2‐aminostrychnine affinity columns. Both isoforms co‐purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.

Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor

Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation.

Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex

Abstract: The inhibitory glycine receptor of mammalian spinal cord is a ligand‐gated chloride channel that, on affinity purification, contains two subunits of 48‐kilodalton (kD) and 58‐kD molecular mass in addition to an associated 93‐kD protein. Ligand‐binding 48‐kD subunit and 93‐kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit ≤ 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93‐kD protein was found to co‐distribute at a fixed stoichiometry with the 48‐kD subunit throughout the CNS of the rat. Moreover, the 93‐kD protein cofractionated with the ligand‐binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93‐kD polypeptide is selectively associated with the membrane core of the strychnine‐sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co‐purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide‐dependent kinases, calcium/calmodulin‐dependent kinases, or protein kinase C. A five‐fold stimulation of kinase activity was, however, observed in the presence of poly‐lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.

Structure and expression of inhibitory glycine receptors.

Signal transmission at chemical synapses involves specific receptors that transduce neurotransmitter binding into alterations of membrane potential. Receptors containing integral ion channels mediate rapid (in the ≤ msec range) transduction events, whereas receptors activating G-protein coupled channels operate at slower time scales (in the msec to sec range). At resting membrane potential, excitation is generated by cation influx, but inhibition of neuronal firing results from increased chloride permeability.

Modulation by NMDA Receptor Antagonists of Glycine Receptor Isoform Expression in Cultured Spinal Cord Neurons

Two developmentally regulated isoforms of the inhibitory glycine receptor harbouring different α subunit variants, GlyRN (neonatal) and GlyRA (adult), have previously been identified in rodent spinal cord. Primary cultures of embyronic spinal neurons, however, express predominantly GlyRN. Here, N‐methyl‐d‐aspartate (NMDA) receptor antagonists were found to significantly increase glycine receptor levels in mouse spinal cord cultures. In the presence of 2‐amino‐5‐phosphonovalerate or MK‐801 (dizocilpine), both GlyRN and GlyRA contents were elevated, as revealed by isoform‐selective immunoassays and amplification of corresponding α subunit transcripts by the polymerase chain reaction. This effect of NMDA receptor antagonists was restricted to a ‘sensitive’ period within the second week after plating. Apparently, NMDA receptor‐mediated glutamate neurotoxicity prevented GlyRA accumulation under standard culture conditions. Our data indicate that neuronal maturation in cell culture depends on conditions which minimize cell death resulting from glutamate release into the culture medium.

Glycine receptor regulation in rodent spinal cord

The inhibitory action of glycine on spinal cord neurons is effectively antagonized by the convulsant alkaloid strychnine. Conversely, glycine-displaceable [3H]strychnine binding has been widely used as a reliable assay for the postsynaptic glycine receptor [GlyR] (Young and Snyder, 1973, 1974). A single class of high-affinity [3H]strychnine binding sites is found with affinity constants of 3–10 nM (for review, see Betz and Becker, 1988). Affinity-purified GlyR contains three polypeptides of 48 kD, 58 kD, and 93 kD molecular weight (Pfeiffer et al., 1982; Graham et al., 1985; Becker et al., 1986; see also Langosch et al., this volume). The primary structure of the ligand-binding 48 kD subunit has been determined by cDNA cloning (Grenningloh et al., 1987). Both the 48 kD and 58 kD polypeptides are integral membrane proteins, whereas the 93 kD polypeptide is a peripheral membrane component (Schmitt et al., 1987) found at the cytoplasmic face of the postsynaptic membrane (Triller et al., 1987).

Immunological identification of a neonatal glycine receptor isotype and its expression in primary cultures of spinal cord

Development of chemosensitivity is a major focus of interest in neurobiology. During formation of the mammalian neuromuscular junction, the embryonic subtype of the nicotinic acetylcholine receptor is replaced by an adult receptor type. This process coincides with a redistribution of receptors from extrasynaptic to subsynaptic areas of the muscle cell membrane (for reviews, see Schuetze and Role, 1987; Brehm et al., 1988). It is not known whether, in the CNS, synaptogenesis is also accompanied by changes in receptor subtype expression and distribution. So far, studies have been hampered by low abundance of these proteins and lack of suitable methodology.