Research Focus
Synaptic and Tonic Inhibition – Glycine Receptor Dynamics from the Point of View of Gephyrin
Neurotransmitter receptors are highly mobile entities of the neuronal plasma membrane. Enrichment of postsynaptic domains with neurotransmitter receptors therefore reflects a dynamic equilibrium between less mobile synaptic and highly mobile non-synaptic receptors (Nature Neuroscience 4:253, 2001). The diffusion rate is slowed by reversible glycine receptor binding to the postsynaptic scaffolding protein gephyrin. These receptors contribute to synaptic inhibition of action potential generation whereas highly mobile receptors, which escape postsynaptic anchoring, are involved in tonic inhibition of neuron firing. We have identified a novel gephyrin splice variant (Molecular and Cellular Neuroscience 16:566, 2000) that adopts specific functions in the hippocampus. This splice variant contains exon 6 (for GPHN gene structure see Journal Biological Chemistry 281:34918, 2006) and confines postsynaptic glycine receptor anchoring at GABAergic synapses (Journal of Neuroscience 24:1398, 2004), thus favoring the occurrence of non-synaptic receptors (European Journal of Neuroscience 30:1077, 2009).
RNA Plasticity in Hyperexcitability Disorders
We have recently isolated mRNAs coding for gain-of-function glycine receptors with substantially increased apparent affinities for glycine and taurine, rendering them well adapted for tonic inhibition in response to hippocampal extracellular glycine. Tonic inhibition of neuron firing plays a pivotal role in brain information transfer as it provides a global control of neuronal excitability (Frontiers in Molecular Neuroscience 1:2, 2008). A large body of evidence has implicated impaired hippocampal GABAergic inhibition in enhanced susceptibility of neurons to become hyperexcitable and to generate epileptiform discharges. Therefore, gain-of-function glycine receptors could help control excitability through tonic inhibition of neurons with impaired GABAergic inhibition. In fact, activation of hippocampal glycine receptors through blockade of cellular glycine uptake was shown to be anticonvulsive (Neuropsychopharmacology 33:701, 2008).
We could establish that these high affinity glycine receptors arise from post-transcriptional C-to-U RNA editing, as demonstrated by the absence of respective genomic sequences and by our finding that blockade of C-to-U RNA editing with the cytidine deaminase transition state inhibitor Zebularine is effective on tonic glycinergic currents (Nature Neuroscience 8:736, 2005). C-to-U editing of GLRA1-3 transcripts results in proline-to-leucine amino acid substitution in the ligand binding domain. As this single amino acid substitution renders all α-subunit glycine receptors high affinity receptors we have identified a unique position within the ~ 430 amino acid glycine receptor protein (Frontiers in Molecular Neuroscience 2:23, 2009). Furthermore, glycine receptor RNA editing is conserved across species and hence also occurs in humans (Journal of Cellular and Molecular Medicine 12:2848, 2008).
Functional analysis at a cellular level, using selective high affinity receptor activation in primary hippocampal neurons, and at a systemic level, using high affinity receptor screening of resected hippocampi from mesial temporal lobe epilepsy patients points to a compensatory and homeostatic, but pathophysiological, role of high affinity glycine receptors. Conversely, in the hippocampus of healthy individuals high affinity glycine receptors are almost absent (Journal of Cellular and Molecular Medicine 12:2848, 2008). Therefore, we are interested in developing these disease-specific roles of high affinity glycine receptors into novel pharmacological approaches to medication of hyperexcitability disorders.
Furthermore, it has become evident that cellular stress can cause dysfunction of the RNA splice machinery. Intracellular alkalosis or high temperature lead to skipping of exons in gephyrin mRNA, which inhibits recruitment of gephyrin as well as GABA(A) receptors to postsynaptic domains, where they are required to handle excitation (Brain 133:3778-3794, 2010). Thus, it appears as if mutability of gene transcripts is involved in the development of hyperexcitability disorders, which might provide an explanation for the discrepancy between the high number of patients and relatively few cases with identified genetic defects.
Deciphering the Molecular Basis of Molybdenum Cofactor Biosynthesis
Besides its pivotal role in inhibitory synaptic transmission, gephyrin has enzymatic activity. It is a multidomain protein that emerged from fusion of two bacterial proteins, MogA and MoeA. These Escherichia Coli proteins contribute to biosynthesis of molybdenum cofactor (Moco), an essential component of cellular redox reactions. Mammalian gephyrins are still able to synthesize Moco because the enzymatic activity of the E. Coli homologous domains is preserved. In mammals, the most important Molybdenum enzyme is sulfite oxidase, which catalyzes the last step in the degradation of sulfur-containing amino acids and sulfatides. Human Moco deficiency is a hereditary metabolic disorder characterized by severe neurodegeneration and resulting in severe epilepsy and early childhood death, which can be prevented through delivery of metabolic precursors. We have found gephyrins with exon 6 to be enzymatically inactive (Journal of Biological Chemistry 283:17370, 2008). Therefore, the decision of a cell to include exon 6 has an impact on regulation of the Moco content in liver and brain. In the brain, non-neuronal cells are the main sources of Moco (Journal of Biological Chemistry 283:17370, 2008). Therefore, another aspect of our work concerns the molecular and functional dissection of posttranscriptional gephyrin processing in a variety of cell types.