GLRB allelic variation associated with agoraphobic cognitions, increased startle response and fear network activation: a potential neurogenetic pathway to panic disorder.

The molecular genetics of panic disorder (PD) with and without agoraphobia (AG) are still largely unknown and progress is hampered by small sample sizes. We therefore performed a genome-wide association study with a dimensional, PD/AG-related anxiety phenotype based on the Agoraphobia Cognition Questionnaire (ACQ) in a sample of 1370 healthy German volunteers of the CRC TRR58 MEGA study wave 1. A genome-wide significant association was found between ACQ and single non-coding nucleotide variants of the GLRB gene (rs78726293, P=3.3 × 10-8; rs191260602, P=3.9 × 10-8). We followed up on this finding in a larger dimensional ACQ sample (N=2547) and in independent samples with a dichotomous AG phenotype based on the Symptoms Checklist (SCL-90; N=3845) and a case-control sample with the categorical phenotype PD/AG (Ncombined =1012) obtaining highly significant P-values also for GLRB single-nucleotide variants rs17035816 (P=3.8 × 10-4) and rs7688285 (P=7.6 × 10-5). GLRB gene expression was found to be modulated by rs7688285 in brain tissue, as well as cell culture. Analyses of intermediate PD/AG phenotypes demonstrated increased startle reflex and increased fear network, as well as general sensory activation by GLRB risk gene variants rs78726293, rs191260602, rs17035816 and rs7688285. Partial Glrb knockout mice demonstrated an agoraphobic phenotype. In conjunction with the clinical observation that rare coding GLRB gene mutations are associated with the neurological disorder hyperekplexia characterized by a generalized startle reaction and agoraphobic behavior, our data provide evidence that non-coding, although functional GLRB gene polymorphisms may predispose to PD by increasing startle response and agoraphobic cognitions.

Autoimmunity to gephyrin in Stiff-Man syndrome.

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by chronic rigidity, spasms, and autoimmunity directed against synaptic antigens, most often the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In a subset of cases, SMS has an autoimmune paraneoplastic origin. We report here the identification of high-titer autoantibodies directed against gephyrin in a patient with clinical features of SMS and mediastinal cancer. Gephyrin is a cytosolic protein selectively concentrated at the postsynaptic membrane of inhibitory synapses, where it is associated with GABA(A) and glycine receptors. Our findings provide new evidence for a close link between autoimmunity directed against components of inhibitory synapses and neurological conditions characterized by chronic rigidity and spasms.

Identification of domains and amino acids involved in GLuR7 ion channel function.

The kainate receptors GluR6 and GluR7 differ considerably in their ion channel properties, despite sharing 86% amino acid sequence identity. When expressed in Xenopus oocytes GluR6 conducts large agonist-evoked currents, whereas GluR7 lacks measurable currents. In the present study, we localized the determinants that are responsible for the functional differences between GluR6 and GluR7 to the extracellular loop domain L3. In addition, we generated several GluR7 point mutants that are able to conduct currents that can be readily measured in Xenopus oocytes. In GluR6, glutamate- and kainate-evoked maximal currents are of the same magnitude when desensitization is inhibited with the lectin concanavalin A. By contrast, all functional GluR7 mutants were found to have glutamate current amplitudes significantly larger than those evoked by kainate. We localized the domain that determines the relative agonist efficacies to the C-terminal half of the L3 domain of GluR7. Our data show that EC(50) values for glutamate (but not for kainate) in GluR7 mutants or chimeras tend to be increased in comparison to the EC(50) values in GluR6. The high EC(50) for wild-type GluR7 reported in the literature appears to be linked to the S1 portion of the agonist-binding domain. Finally, we determined the C-terminal half of the L3 domain plus the far C-terminal domain of GluR7 to be responsible for the recently reported reduction of current amplitude seen when GluR7 is coexpressed with GluR6. We conclude that coexpression of GluR6 and GluR7 leads to nonstochastical assembly of heteromeric receptor complexes.

Length of the TM3-4 loop of the glycine receptor modulates receptor desensitization.

Recent studies on the molecular determinants important for glycine receptor biogenesis and function mechanisms indicate an important role of basic residues within the intracellular loop between transmembrane domains (TM) 3 and 4. We investigate the role of loop length and loop exchange in combination with the presence or absence of basic stretches (318)RRKRR and (385)KKIDK of the human glycine receptor α1 using expression in transfected cell lines. Exchanges of the large intracellular loop between members of the Cys-loop receptor family have been shown to keep functionality of the host receptor. Here, constructs were generated with deletion of the intracellular loop of the glycine receptor α1, insertion of the loop from the prokaryotic Cys-loop receptor of Gloeobacter violaceus both with and without leaving the basic stretches at the N-terminal and C-terminal part of the intracellular domain. All receptor constructs were expressed at the cell surface with the significantly lowest expression of the construct with a deletion of the glycine receptor α1 TM3-4 loop, except the two basic stretches adjoined. Functionality of the inhibitory glycine receptor chimeras was demonstrated with whole cell recordings from transfected cells. Chimeras lacking the basic stretches result in non-functionality. An analysis of receptor desensitization demonstrated that close proximity of both basic stretches resulted in large fractions of desensitizing currents. We conclude that the TM3-4 loop length is critical for glycine receptor α1 desensitization and a direct neighborhood of both basic stretches changes receptor properties from non-desensitizing to desensitizing.

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons.

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.

N-Glycosylation is not a prerequisite for glutamate receptor function but Is essential for lectin modulation.

All ionotropic glutamate receptor (iGluR) subunits analyzed so far are heavily N-glycosylated at multiple sites on their amino-terminal extracellular domains. Although the exact functional significance of this glycosylation remains to be determined, it has been suggested that N-glycosylation may be a precondition for the formation of functional ion channels. In particular, it has been argued that N-glycosylation is required for the formation of functional ligand binding sites. We analyzed heterologously expressed recombinant glutamate receptors (GluRs) of all three pharmacological subclasses of glutamate receptors, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and kainate receptors. By expressing the GluR subunits in tunicamycin-treated, nonglycosylating Xenopus laevis oocytes, we determined that in neither case is N-glycosylation required for ion channel function, although for NMDA receptors, functional expression in the absence of N-glycosylation is very low. Furthermore, we analyzed and compared the interaction of the desensitization-inhibiting lectin concanavalin A (ConA) with all functional GluR subunits. We show that although ConA has its most pronounced effects on kainate receptors, it potentiates currents at most other receptor subtypes as well, including certain NMDA receptor subunits, although to a much lesser extent. One notable exception is the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2, which is not affected by ConA. Furthermore, we show that ConA acts directly via binding to the carbohydrate side chains of the receptor protein.

Kainate binding proteins possess functional ion channel domains.

Kainate binding proteins (KBPs) are highly homologous to ionotropic glutamate receptors; however, no ion channel function has been demonstrated for these proteins. To investigate possible reasons for the apparent lack of ion channel function we transplanted the ion channel domains of five KBPs into glutamate receptors GluR 6 and GluR1. In each case we obtained functional chimeric receptors in which glutamatergic agonists were able to open the KBP-derived ion channel with EC50 values identical to those of the subunit contributing the ligand binding domain. Maximal current amplitudes were significantly smaller than those of the parent clones, however. We also show that the KBP ion channels are highly permeable for calcium and have certain pharmacological properties that are distinct from all other glutamate receptor (GluR) subunits. Thus, all five known KBPs, in addition to their well characterized functional ligand binding sites, have functional ion permeation pathways. Our data suggest that the lack of ion channel function in wild-type KBPs results from a failure to translate ligand binding into channel opening. We interpret our findings to indicate the requirement for a modulatory protein or an additional subunit serving to alter the structure of the KBP subunit complex such that signal transduction is enabled from the ligand binding site to the intrinsically functional ion pore.

Investigation by ion channel domain transplantation of rat glutamate receptor subunits, orphan receptors and a putative NMDA receptor subunit.

Among the 18 ionotropic glutamate receptor subunits identified in the mammalian central nervous system, five (delta1, delta2, GluR7, chi2 and NR3A, formerly called NMDAR-L or chi1) reportedly fail to form functional ion channels in heterologous expression systems. Four of these subunits, delta1, delta2, chi2 and NR3A, have not even been shown to bind glutamatergic ligands, relegating them to the status of 'orphan' receptors. We used a domain transplantation approach to investigate potential functional properties of the putative ion channel domains of four of these subunits. By exchanging ion pore domains between functional glutamate receptors (GluR1, GluR6 and NMDAR1) with known pore properties we first tested the feasibility of the domain swapping method. We demonstrate that ion channel domains can be transplanted between all three functional subfamilies of ionotropic glutamate receptors. Furthermore, exchange of ion pore domains allows identification of those channel properties determined exclusively by the ion pore. We then show that transplanting the pore domain of GluR7 into either GluR1 or GluR6 generates perfectly functional ligand-gated ion channels that allow characterization of electrophysiological and pharmacological properties of the GluR7 pore domain. In contrast, delta1, delta2 and NR3A do not produce functional receptors when their pore domains are transplanted into either the AMPA receptor, GluR1, the kainate receptor, GluR6, or the NMDA receptor, NMDAR1. We speculate that the orphan receptors delta1 and delta2, and the NMDA receptor-like subunit NR3A may serve some modulatory function, rather than contributing to the formation of ion channels.

Computer-aided modeling of structure stabilizing disulfide bonds in recombinant human interferon-gamma.

We present a general search algorithm for possible insertion sites of disulfide bonds in proteins based on the coordinates of the solved X-ray or NMR structure, allowing the insertion of disulfide bonds with a minimum of conformational tension and backbone rearrangements. The FORTRAN 77 program "Ssuitable' was written for this purpose. This methodological approach was applied to recombinant human interferon-gamma (rhu-IFN-gamma), a cytokine of great pharmaceutical interest with a wide variety of biological activities including antiviral, antiproliferative and immunomodulatory effects. A model based on the C alpha-coordinates obtained from the Brookhaven data base was built. Four different insertion sites were selected in the model, connecting the two subunits of the homodimer. The thermodynamic stability of rhu-IFN-gamma is low, limiting its clinical application. We expect that the insertion of additional new disulfide bonds will enhance the thermodynamic stability as well as protect the protein against proteolytic degradation.

Engineered disulfide bonds in recombinant human interferon-gamma: the impact of the N-terminal helix A and the AB-loop on protein stability.

Insertion sites for cysteines with optimal stereochemistry for the formation of unstrained disulfide bridges were identified in recombinant human interferon-gamma (rhu-IFN-gamma) by computer modelling. We have engineered two different disulfide cross-linked mutants, containing a pair of symmetry-related disulfide bonds, which stabilize the N-termini of both monomers of the homodimeric protein. Mutations E7C and S69C allow the formation of an intramonomer disulfide bond between helices A and D. In contrast, the A17C and H111C mutations lead to a covalent cross-link between both monomers. The AB-loop is linked to helix F. The fluorescence properties of native and disulfide cross-linked proteins were studied as a function of guanidine hydrochloride concentration. Melting temperatures (Tm) were calculated from the decrease in CD ellipticity at 220 nm. The induction of the antiviral effect was measured using A549 fibroblast cells infected with encephalomyocarditis virus. The ability to induce the expression of the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activated cell scanning analysis. The stability of both mutants was strongly enhanced against temperature- and cosolvent-induced unfolding. The delta Tm of mutant IFN-gamma E7C/S69C was 15 degrees C. All measured biological activities of this mutant were equal to wild type. In the case of the other mutant IFN-gamma A17C/H111C, the delta Tm value was 25 degrees C. This mutation abolishes nearly the entire biological activity (< 1%) with no detectable changes of secondary structure in the CD spectrum. Our results illustrate the importance of the N-terminal helix A and the AB-loop for the unfolding pathway and thermodynamic stability of rhu-IFN-gamma.

Interferon-gamma variants with deletions in the AB surface loop--flexibility is a critical point for receptor binding.

The receptor-binding AB loop of recombinant human interferon-gamma (IFN-gamma) has multiple contacts with the extracellular part of the IFN-gamma receptor a chain (IFN-gammaRalpha). We explored the possible length of truncated AB loops and their conformations by molecular modelling. Deletions of two amino acids at the tip of the loop were tolerated in the model without van der Waals collisions of the AB loop with helix F. Based on these modelling results, two deletion mutants were constructed by overlap-extension PCR mutagenesis: des-(A23, D24)-IFN-gamma and des-(N25, G26)-IFN-gamma. Both mutations were tolerated by the folding pattern of recombinant human IFN-gamma, as proved by CD spectroscopy. The stability of both mutants against cosolvent-induced unfolding was equal to that of wild-type IFN-gamma. In contrast to the biophysical similarities of wild-type and mutant IFN-gamma proteins, the biological activities of both mutants dropped significantly. Antiviral activity and human leucocyte antigen (HLA)-DR induction of des-(N25, G26)-IFN-gamma was 10% that of wild-type activity. des-(A23, D24)-IFN-gamma had only 1% remaining activity. Receptor-binding experiments confirmed that both deletions had a negative influence on the affinity of recombinant human IFN-gamma to its cellular receptor. We conclude from this combined molecular modelling and mutagenesis experiments, that the reduced flexibility of the truncated AB loop abrogates the possibility of the formation of a 3(10) helix in the receptor-bound state as observed in the X-ray structure of the IFN-gammaRalpha-IFN-gamma complex.

Opposing effects of molecular volume and charge at the hyperekplexia site alpha 1(P250) govern glycine receptor activation and desensitization.

Allelic variants of the glycine receptor alpha1 subunit gene GLRA1 underlie the human neurological disorder hyperekplexia. Among these, the subunit variant alpha1(P250T) is characterized by an amino acid substitution within the cytoplasmic TM1-2 loop. To identify structural elements at position alpha1(250) that govern receptor function, homomeric mutant receptor channels were subjected to electrophysiological analysis after recombinant expression in HEK293 cells. Wild-type alpha1(P250) channels were nondesensitizing with an EC(50) for glycine of 8 microm, whereas bulky hydrophobic side chains of the channel variants alpha1(P250V/I/L/F) showed rapid desensitization (tau(desens), 50-250 ms) and EC(50) values of 400-1800 microm. Small side chains (P250G/A/S) gave rise to wild-type-like channels. Effects of volume were counteracted by charge: alpha1(P250E/R) were nondesensitizing; EC(50) was approximately 70 microm. The mutants alpha1(P250C/Y) displayed intermediate channel properties (EC(50), 42/70 microm; tau(desens), 3300/2800 ms, respectively). The isotropic forces volume and hydropathy were sufficient to account for the observed effects of residue alpha1(250) on receptor function. Indeed, channel behavior was best predicted by a combined hydropathy/volume index describing the hydrophobic surface of individual amino acids. These observations characterize the short intracellular TM1-2 loop as a regulatory domain for channel activation and a crucial mediator of glycine receptor desensitization.