Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) ␣1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR ␣1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/␤2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR ␣1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/␤2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.

Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. coli homologue, DnaK, which together suggest that HscC is a specialized chaperone. The basal ATPase cycle of HscC had k cat and K m values that were 8-and 10,000-fold higher than for DnaK. The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested. Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK. These differences are explicable by distinct structural variations. HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate. In vivo, HscC failed to complement temperature sensitivity of ⌬dnaK cells. Deletion of hscC caused a slow growth phenotype that was suppressed after several generations. Triple knock-outs of all E. coli genes encoding Hsp70 proteins (⌬dnaK ⌬hscA ⌬hscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability. An extensive search for ⌬hscC phenotypes revealed a hypersensitivity to Cd 2؉ ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments. Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function.

The strychnine-sensitive glycine receptor (GlyR) is a ligandgated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyR␣1-4 and -␤). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyR␣1 and -␣3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and ␤-galactosidase-tagged domain constructs, the TM3-4 loops of GlyR␣1 and -␣3, but not of GlyR␣2 and -␤, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin ␣3 and ␣4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.In the vertebrate nervous system, signal transmission at chemical synapses is mediated by ionotropic and metabotropic neurotransmitter receptors. Ligand-gated ion channels harbor an intrinsic channel pore that is opened almost instantly upon ligand binding. Among the ligand-gated ion channels, the superfamily of Cys-loop receptors comprises the nicotinic acetylcholine receptor, the 5-hydroxytryptamine type 3 receptor, the ␥-aminobutyric acid type A/C receptor, and the GlyR. Besides sequence homology, Cys-loop receptors share a common pentameric rosette-like composition of homologous subunits and a characteristic subunit topology (Fig.

Objective Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff‐person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood. Methods A cell‐based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model. Results Glycine receptor function as assessed by glycine dose–response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N‐terminal residues 29A to 62G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals. Interpretation Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff‐person syndrome or progressive encephalitis with rigidity and myoclonus patients. ANN NEUROL 2020;88:544–561

Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations. AbbreviationsCLR, cys-loop receptor; ECD, extracellular domain; GEFS+, generalized epilepsy with febrile seizures plus; Gly, glycine; M1-M4, transmembrane domains 1-4; PTX, picrotoxin History of human hyperekplexiaThe ability of strychnine to influence inhibitory reflexes and to convert these into excitatory reflexes was originally shown by Owen and Sherrington (1911). Hyperekplexia (Startle disease, Stiff baby syndrome, STHE -startle disease or hyperekplexia, OMIM 149400) was first described by Kirstein and Silfverskiold (1958) long before recombinant systems became available. In 1966 Suhren et al. reported a family with 25 members through five generations that suffered from abnormal severe startle reactions. This family was clinically described as having an autosomal dominant inheritance with an abnormal startle reaction in affected patients, which was elicited by different stimuli that did not provoking similar reactions in healthy controls. With the help of electroencephalographic (EEG) observations, a subcortical origin, for example, some midline structure like the brainstem, was proposed as being the source for this abnormal startle reaction. Furthermore, the patients were c...