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Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

In the present study we have characterized the biophysical properties of wild-type (WT) α1β2 and α3β2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1β2 and α3β2 activation differed significantly, with α1β2 and α3β2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1β2 and α3β2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αβ2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDβ2 and α3ECD/α1TMDβ2 receptors were intermediate to those of WT α1β2 and WT α3β2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDβ2 (23 ± 3%) was similar to that of α3β2 but significantly different from that of α1β2, whereas the residual current displayed by α3ECD/α1TMDβ2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1β2 and α3β2 receptors.

Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycine-gated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level.

Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC50 of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3–6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.

Ligand-binding of Cys-loop receptors results in rearrangements of extracellular loop structures which are further translated into the tilting of membrane spanning helices, and finally opening of the ion channels. The cryo-EM structure of the homopentameric α1 glycine receptor (GlyR) demonstrated an involvement of the extracellular β8–β9 loop in the transition from ligand-bound receptors to the open channel state. Recently, we identified a functional role of the β8–β9 loop in a novel startle disease mouse model shaky. The mutation of residue GlyRα1Q177 to lysine present in shaky mice resulted in reduced glycine potency, reduced synaptic expression, and a disrupted hydrogen network at the structural level around position GlyRα1Q177. Here, we investigated the role of amino acid volume, side chain length, and charge at position Q177 to get deeper insights into the functional role of the β8–β9 loop. We used a combined approach of in vitro expression analysis, functional electrophysiological recordings, and GlyR modeling to describe the role of Q177 for GlyR ion channel function. GlyRα1Q177 variants do not disturb ion channel transport to the cellular surface of transfected cells, neither in homomeric nor in heteromeric GlyR configurations. The EC50 values were increased for all GlyRα1Q177 variants in comparison to the wild type. The largest decrease in glycine potency was observed for the variant GlyRα1Q177R. Potencies of the partial agonists β-alanine and taurine were also reduced. Our data are further supported by homology modeling. The GlyRα1Q177R variant does not form hydrogen bonds with the surrounding network of residue Q177 similar to the substitution with a basic lysine present in the mouse mutant shaky. Among all investigated Q177 mutants, the neutral exchange of glutamine to asparagine as well as the introduction of the closely related amino acid glutamic acid preserve the hydrogen bond network. Introduction of amino acids with small side chains or larger volume resulted in a loss of their hydrogen bonds to neighboring residues. The β8–β9 loop is thus an important structural and functional determinant of the inhibitory GlyR.

Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABAA receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC10−30 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.