Volker Schmieden

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine‐gated strychnine‐sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent‐human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2‐p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.

Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors.

1. The effect of the divalent cation Zn2+ on inhibitory glycine receptor (GlyR) currents was investigated in rat embryonic spinal cord neurons and Xenopus oocytes expressing recombinant GlyRs. 2. In cultured spinal neurons, Zn2+ potentiated glycine‐induced whole‐cell currents about 3‐fold when applied extracellularly at concentrations of 0.5‐10 microM. In contrast, higher concentrations (> 100 microM) of Zn2+ decreased the glycine response. 3. A similar biphasic modulation of glycine‐induced currents by Zn2+ was also found with recombinant homo‐ and hetero‐oligomeric GlyRs generated in Xenopus oocytes. Dose‐response analysis showed that both the potentiating and inhibitory effects of Zn2+ result from changes in apparent agonist affinity. 4. Analysis of chimeric constructs of the GlyR alpha 1‐ and beta‐subunits revealed that the positive and negative modulatory effects of Zn2+ are mediated by different regions of the alpha 1‐subunit. 5. Our data indicate the existence of distinct high‐ and low‐affinity Zn2+ binding sites on the ligand‐binding alpha‐subunits of the GlyR. These sites may be implicated in the regulation of synaptic efficacy within glycinergic pathways.

A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit

Agonist activation of the inhibitory glycine receptor (GlyR) in the adult vertebrate CNS is efficiently antagonized by the alkaloid strychnine. Here, we describe a novel rat GlyR alpha subunit cDNA (alpha 2*) that generates chloride channels of low strychnine sensitivity upon expression in Xenopus oocytes. Comparison with the highly homologous human alpha 2 polypeptide and site-directed mutagenesis identified a single amino acid exchange at position 167 that causes the altered pharmacology of alpha 2* receptors. Amplification by the polymerase chain reaction revealed a strong decrease in alpha 2* mRNA abundancy during postnatal spinal cord development. These data indicate that alpha 2* represents a ligand binding subunit of the previously identified neonatal GlyR isoform of low strychnine affinity.

Agonist pharmacology of neonatal and adult glycine receptor alpha subunits: identification of amino acid residues involved in taurine activation.

The inhibitory glycine receptor (GlyR) is a pentameric chloride channel protein which mediates postsynaptic inhibition in the mammalian central nervous system. In spinal cord, different GlyR isoforms originate from the sequential expression of developmentally regulated variants of the ligand binding alpha subunit. Here, neonatal alpha 2 and adult alpha 1 subunits are shown to generate GlyRs with distinct agonist activation profiles upon heterologous expression in Xenopus oocytes. Whereas alpha 1 receptors are efficiently gated by beta‐alanine and taurine, alpha 2 GlyRs show only a low relative response to these agonists, which also display a reduced sensitivity to inhibition by the glycinergic antagonist strychnine. Construction of an alpha 2/alpha 1 subunit chimera and site‐directed mutagenesis of the extracellular region of the alpha 1 sequence identified amino acid positions 111 and 212 as important determinants of taurine activation. Our results indicate the existence of distinct subsites for agonists on alpha 1 and alpha 2 GlyRs and suggest that the ligand binding pocket of these receptor proteins is formed from discontinuous domains of their extracellular region.

Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia.

Hereditary hyperekplexia is a dominant neurological disorder associated with point mutations at the channel‐forming segment M2 of the glycine receptor alpha 1 subunit. Voltage‐clamp recordings from the heterologously expressed mutants (alpha 1R271L or alpha 1R271Q) revealed 146‐ to 183‐fold decreased potencies of glycine to activate the chloride channel, and significantly reduced maximal whole‐cell currents as compared with wild‐type receptors. In contrast, the ability of the competitive antagonist strychnine to block glycine‐induced currents was similar in all cases. Radioligand binding assays showed a 90‐ to 1365‐fold reduction in the ability of glycine to displace [3H]strychnine from its binding site on the mutant receptors. Paralleling the reductions in whole‐cell current, the elementary main‐state conductances of the mutants (alpha 1R271L, 64 pS; alpha 1R271Q, 14 pS) were lower than that of the wild‐type receptor (86 pS). The decreased agonist affinities and chloride conductances of the mutants are likely to cause neural hyperexcitability of affected patients by impairing glycinergic inhibition. In addition, our data reveal that structural modifications of the ion‐channel region can affect agonist binding to the glycine receptor.

Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor

The inhibitory postsynaptic glycine receptor (GlyR) of rat spinal cord is an oligomeric transmembrane protein which forms an agonist‐gated anion channel. Expression in Xenopus oocytes of its mol. wt 48,000 subunit generated glycine‐gated chloride channels which were analysed by voltage clamp. The agonist and antagonist response properties as well as the desensitization characteristics of these 48 kd subunit receptors resembled GlyRs expressed from spinal cord poly(A)+ RNA. These data indicate that the 48 kd subunit is capable of assembling into a functional receptor homo‐oligomer which displays the pharmacology characteristic of the spinal cord GlyR.

Alternative splicing generates two isoforms of the α2 subunit of the inhibitory glycine receptor

The inhibitory glycine receptor (GlyR) is a ligand‐gated chloride channel protein which displays developmental heterogeneity in the mammalian central nervous system. Here we describe 2 novel cDNA variants of the rat GlyR α2 subunit and demonstrate that alternative splicing generates these 2 isoforms. The deduced protein sequences (α2A and α2B) exhibit 99% identity with the previously characterized human α2 subunit. In situ hybridization revealed expression of both α2A und α2B mRNAs in the prenatal rat brain, suggesting that these variant proteins may have a role in synaptogenesis. Heterologous expression in Xenopus oocytes showed that the more abundantly expressed α2A subunit forms strychnine‐sensitive ion channels which resemble human α2 subunit GlyRs in their electrophysiological properties.

Structure and Functions of Inhibitory and Excitatory Glycine Receptors

ABSTRACT: The strychnine‐sensitive glycine receptor (GlyR) is a pentameric chloride channel protein that exists in several developmentally and regionally regulated isoforms in the CNS. These result from the differential expression of four genes encoding different variants (α1‐α4) of the ligand‐binding subunit of the GlyR. Their assembly with the structural b subunit is governed by “assembly cassettes” within the extracellular domains of these proteins and creates chloride channels of distinct conductance properties. GlyR gating is potentiated by Zn2+, a metal ion co‐released with different neurotransmitters. Site‐directed mutagenesis has unraveled major determinants of agonist binding and Zn2+ potentiation. During development, glycine receptors mediate excitation that results in Ca2+ influx and neurotransmitter release. Ca2+ influx triggered by the activation of embryonic GlyRs is required for the synaptic localization of the GlyR and its anchoring protein gepyhrin. In the adult, mutations in GlyR subunit genes result in motor disorders. The spastic and spasmodic phenotypes in mouse as well as human hereditary startle disease will be discussed.

Point mutation of glycine receptor α1 subunit in the spasmodic mouse affects agonist responses

Homozygotic spasmodic (spd/spd) mice suffer from a motor disorder resembling poisoning by the glycine receptor antagonist strychnine. Here, a point mutation was identified in the glycine receptor α1 subunit gene of the spasmodic mouse which predicts an alanine‐to‐serine exchange at position 52 of the mature polypeptide. Upon expression in Xenopus laevis oocytes, α1A52S receptor channels displayed reduced responses to glycine, β‐alanine and taurine when compared to recombinant α1 glycine receptors. As glycine receptor content in spinal cord and native molecular weight appeared unaltered, this suggests that the spasmodic phenotype results from an altered neurotransmitter sensitivity of the mutant α1A52S subunit.

Isoform-selective deficit of glycine receptors in the mouse mutant spastic

The mutant mouse spastic (spa) develops a characteristic motor disorder about 2 weeks after birth, with symptoms resembling sublethal poisoning by the glycinergic antagonist strychnine. Correspondingly, adult homozygotic mutants (spa/spa) exhibit a severe reduction of inhibitory glycine receptors in spinal cord and brain. Here we show that the spastic mutation selectively interferes with the postnatal accumulation of the adult isoform of the glycine receptor protein, whereas perinatal expression of the neonatal receptor isoform is not detectably affected. Heterologous expression in X. laevis oocytes of poly(A)+ RNA and Northern blot analysis indicate normal levels of glycine receptor alpha 1 subunit transcripts in spinal cord of adult spastic mutants. Thus, the age-dependent manifestation of spastic symptoms after birth reflects a selective effect of the mutation on the developmental expression of the adult glycine receptor isoform.