The neurotransmitter receptor for glycine (GlyR) is a ligand-gated chloride-permeable ion channel in the central nervous system, and dysregulation of this channel is associated with diverse neurological disorders. Cesium (Cs+) is an agonist of GlyRs, and recent atomistic molecular dynamic simulations suggested amino acids D141, E192, and D194 with negatively charged side chains as possible binding sites for Cs+. To test this hypothesis, we mutated these positions to code for alanine with a neutral hydrophobic side chain or lysine with a positively charged side chain. The correspondingly mutated GlyR channels were expressed in HEK293T cells and analyzed using whole-cell patch clamp electrophysiology. The results show that D141 mutations had no considerable effect on GlyR currents, E192K prolonged desensitization and D194 turned out to be critical for general (glycine- and Cs+-dependent) GlyR activation, which was not due to differentially reduced cell surface expression of all the investigated GlyR mutants. Thus, E192 and D194 are critically involved in cell surface GlyR activation by Cs+ and glycine.
The neurotransmitter receptor for glycine (GlyR) is key to regulation of respiration and neuromotor coordination, hearing, neurogenesis, cortical cell migration, cognition and nociception as it is expressed in neurons of the spinal cord, brainstem, cortex, hippocampus and even in non-neuronal cells. Dysregulation of GlyR function can thus result in several diseases including neurological disorders such as autism, epilepsy and chronic inflammatory pain as well as cardiovascular and respiratory dysfunction. Hence, research focusing on the structure-function relationship of GlyRs is required for a better understanding of the diverse causes of associated diseases.
The GlyR is a chloride-permeable receptor and part of the pentameric, ligand-gated Cys-loop ion channel family. Depending on the chloride concentration, GlyR activation results in inhibition or excitation at the synapse. GlyRs consist of five subunits of either α1-4 or together with a β subunit, which functions as anchor to the postsynaptic matrix protein gephyrin. The receptor has N- and C-terminal extracellular domains separated by four transmembrane-spanning (TM) regions. It was proposed that GlyR gating happens via the movement of TM2 and that the flanking loops, such as the F-loop (β8-β9), Cys-loop (β6-β7), β1-β2 loop, and the β10 strand which precedes the TM1 helix, serve as connections from the extracellular domain to the TM2-TM3 linker. In this way, the conformational change after binding of glycine at the interface between two subunits is transitioned to open the internal pore.
We previously investigated the effect of RNA-editing of GlyRs on the potency of the agonists glycine, taurine, and GABA. C-to-U RNA-editing results in amino acid substitution from proline to leucine at position 185 in the mature signal peptide-processed GlyR α1 and α3 proteins while the edited position corresponds to 192 in mature GlyR α2. In all cases, RNA-editing of GlyRs results in a gain-of-function possibly contributing to the diverse neuropsychiatric symptoms of temporal lobe epilepsy.
The recently identified GlyR agonists Cs and NH also showed increased potency at RNA-edited GlyRs. Atomistic molecular dynamic simulations were utilized to predict possible Cs binding domains in the GlyR and revealed two potential sites in the extracellular receptor domain. The first site is found slightly above the glycine binding site and the more prominent site comprises D141, E192, and D194 flanking the F-loop or β8-β9 loop. Interestingly, the RNA-editable position P185 is located within this region. While D141 is part of the Cys-loop that is conserved in the receptor family, E192 and D194 are located on the β-sheet 9. In addition to F159, Y202, T204 and F207 from one subunit and R65 and S129 from the neighboring subunit that directly participate in glycine binding, this loop structure between β-sheets 8 and 9 emerges as critical determinant of the signal transduction after glycine binding.
Divalent cations, such as Zn and Cu, are also known to bind the GlyR and exhibit modulatory effects on glycine-evoked currents. Zn is a biphasic modulator that either inhibits or potentiates GlyR activation depending on its concentration and by binding to two different sites of the receptor. The binding moiety described for its potentiating effect is assumed to consist of E192, D194 and H215. The E192A and D194A mutations in GlyR α1 ablated the potentiating effect of Zn and GlyR D194A had a threefold higher EC, indicating less potency. For E192 the effect was interpreted as more indirect, possibly through perturbation of the backbone structure. However, GlyRs are not directly activated by Zn, and it was originally proposed that its potentiating effect was due to slowing agonist dissociation, and more recently due to intermediate conformations in the gating cycle. In addition, E192 was also found to be part of the Cu binding site, but interestingly Cu only inhibits the glycine-evoked GlyR currents.
In this study, we mutated the positions D141, E192, and D194 to code for the amino acid alanine with a neutral hydrophobic side chain or lysine with a positively charged side chain. Whole-cell patch clamp electrophysiology using transfected HEK293T cells revealed no significant effect of D141 mutations on GlyR activation by Cs or glycine. However, the E192K channel exhibited a prolonged desensitization and D194 mutations strongly reduced the amplitude of both glycine- and Cs-evoked GlyR currents, which was not due to impaired GlyR surface expression. Thus, our study adds molecular details to the complex structure-function relationships in GlyRs as they reveal E192 and D194 on β-sheet 9 as critical determinant of either GlyR desensitization or activation by Cs and glycine.