Jochen Kuhse

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.

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.

Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells.

In adult spinal neurons inhibitory glycine receptors (GlyR) are localized at postsynaptic membrane specializations underlying glycinergic nerve terminals. The peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic GlyR clusters. Here, we coexpressed GlyR polypeptides and gephyrin in 293 cells and observed rerouting of hetero-oligomeric GlyR and its beta, but not of alpha subunits to intracellular gephyrin aggregates. A GlyR chimeric alpha 1/beta protein was also accumulated at these gephyrin aggregates, indicating that colocalization with gephyrin depends on cytoplasmic domains of the beta subunit. gamma-Aminobutyric acid type-A receptor (GABAAR) subunits were not targeted to intracellular gephyrin aggregates with the exception of the GABAAR beta 3 subunit, which partially colocalized with gephyrin. These data show that gephyrin alters the subcellular localization of the GlyR beta and, to some extent, GABAAR beta 3 subunits. Thus, gephyrin-binding subunits might target hetero-oligomeric ion channels to a gephyrin matrix underlying the differentiating postsynaptic membrane.

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Phosphorylation of gephyrin in hippocampal neurons by cyclin-dependent kinase CDK5 at Ser-270 is dependent on collybistin.

Background: Formation of inhibitory synapses in the CNS is dependent on cluster formation of the scaffold protein gephyrin. Results: Knockdown of collybistin and inhibition of cyclin-dependent kinases (CDK1,- 2, and -5) abolished the phosphorylation of gephyrin detected by mAb7a at Ser-270. Conclusion: Gephyrin detected with mAb7a is phosphorylated at Ser-270. Significance: These data suggest a novel view on kinases involved in gephyrin phosphorylation. Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABAA receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrinT276A,S277A revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin 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.

Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons.

Collybistin (Cb) is a brain specific guanine nucleotide exchange factor that interacts with the inhibitory postsynaptic scaffold protein gephyrin. Cb is essential for the postsynaptic clustering of gephyrin and major GABA(A) receptor subtypes during the formation and maintenance of GABAergic synapses in the hippocampus and other areas of the forebrain. In the rat, four distinct splice variants (Cb1, Cb2(SH3-), Cb2(SH3+) and Cb3), have been described, which differ in their C-termini (Cb1-3) and in respect of the SH3-domain that is absent in Cb2(SH3-). In the human brain, only a single isoform (hPEM2) corresponding to Cb3, was found to be expressed. This has been implicated in neurological defects such as hyperekplexia, epilepsy, anxiety, aggression and mental retardation. In this study, we address the functional significance of the differentially spliced Cb isoforms by generating a shRNA-mediated knock-down of endogenous Cb in hippocampal cultured neurons that is subsequently rescued by the expression of distinct Cb isoforms. We found that the Cb knock-down induced impairment in GABAergic neurotransmission could be rescued by the expression of any of the Cb isoforms, independent of their C-termini or the presence of the SH3-domain in the N-terminal region. Thus, the different Cb isoforms all confer basic functionality.

Molecular architecture of glycinergic synapses

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.

Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity

The translation eukaryotic elongation factor 1α (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the α2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-d-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.