The socio-economic burden of H1-antihistamine-refractory chronic spontaneous urticaria in Germany.

BACKGROUND AND STUDY AIM: Data from the AWARE study (A Worldwide Antihistamine-Refractory chronic urticaria patient Evaluation) illustrate a substantial disease burden in German patients with H1-antihistamine (-H1-AH)-refractory chronic spontaneous urticaria (CSU). Detrimental effects on patients' quality of life, poor disease control and impairment in the ability to work and perform other daily activities are reported. Based on these findings, this study aims to quantify the epidemiological and socio-economic burden of H1-AH-refractory CSU in Germany. METHODS: To determine the epidemiological burden of H1-AH-refractory CSU, the age- and gender-specific prevalence of CSU and the proportion of H1-AH-refractory patients in Germany anonymized data from the InGef research database have been used. In a second step, the socio-economic burden in terms of lost numbers of hours in paid and unpaid work was calculated by extrapolating the age- and gender-specific work productivity and activity impairment (WPAI) observed in AWARE to the H1-AH-refractory CSU population in Germany. Finally, productivity losses in paid and unpaid work were monetized using the human capital and the friction cost approach respectively. Moreover, socio-economic burden was calculated depending on symptom control of the patients (measured by urticaria control test [UCT]). RESULTS: In Germany, over 203,000 patients (20 years or older) had H1-AH-refractory CSU in 2018. The avoided lost paid and unpaid work hours attributable to H1-AH-refractory CSU summed up to over 100 million. Overall, the socio-economic burden of H1-AH-refractory CSU in monetary terms was evaluated at € 2.2 billion and the majority of this was due to unpaid work loss. Patients with poor disease control, as indicated by UCT score < 12, were more likely to suffer from high impairment than patients with controlled disease, resulting in a higher socio-economic burden. CONCLUSION: The results of our analyses picture the substantial socio-economic burden of H1-AH-refractory CSU and therefore the tremendous impact it has on daily lives of individuals and society overall.

The Societal Economic Impact of Secukinumab in First-Line Treatment of Moderate to Severe Plaque Psoriasis in Germany: An Open-Cohort Simulation.

OBJECTIVES: Moderate to severe plaque psoriasis is a chronic inflammatory disease. In Germany, guidelines recommend fumaric acid esters (FAEs) as first-line systemic treatment. Despite treatment with FAEs, disease burden remains high in Germany. Secukinumab, a fully human monoclonal antibody, has demonstrated greater efficacy and safety than FAEs in the PRIME trial. The aim of the current study, hence, is to quantify the potential societal economic impact of secukinumab in systemic treatment-naïve patients with moderate to severe plaque psoriasis in Germany. METHODS: We employed a semi-Markov model to capture health gains at an individual level and a dynamic population model to extrapolate the findings in the population of interest. We quantified the health outcomes in two scenarios: (i) patients receiving secukinumab and (ii) patients receiving FAEs. Using estimates on change in work productivity and societal economic parameters, we translated the health outcomes into paid and unpaid economic gains. We used gross value added (GVA) to value these gains and calculated the macroeconomic indirect and induced value-chain effects. RESULTS: Our calculations show that patients treated with secukinumab spend on average 94% of their time in Psoriasis Area and Severity Index (PASI) ≥ 75 state compared with 80% for patients in the FAEs scenario. When assuming that FAEs are the sole comparator to secukinumab, this difference could lead to 4.3 million active hours gained until 2030. These gained hours translate to a total societal economic impact of €308 million till 2030 for the whole patient population in GVA terms. CONCLUSION: This study demonstrated that using secukinumab instead of FAEs in moderate to severe plaque psoriasis could lead to substantial macroeconomic GVA gains.

Biologic Treatment in Combination with Lifestyle Intervention in Moderate to Severe Plaque Psoriasis and Concomitant Metabolic Syndrome: Rationale and Methodology of the METABOLyx Randomized Controlled Clinical Trial.

Inflammatory diseases including psoriasis are associated with metabolic and cardiovascular comorbidities, including obesity and metabolic syndrome. Obesity is associated with greater psoriasis disease severity and reduced response to treatment. Therefore, targeting metabolic comorbidities could improve patients' health status and psoriasis-specific outcomes. METABOLyx is a randomized controlled trial evaluating the combination of a lifestyle intervention program with secukinumab treatment in psoriasis. Here, the rationale, methodology and baseline patient characteristics of METABOLyx are presented. A total of 768 patients with concomitant moderate to severe plaque psoriasis and metabolic syndrome were randomized to secukinumab 300 mg, or secukinumab 300 mg plus a tailored lifestyle intervention program, over 24 weeks. A substudy of immunologic and metabolic biomarkers is ongoing. The primary endpoint of METABOLyx is PASI90 response at week 24. Other endpoints include patient-reported outcomes and safety. METABOLyx represents the first large scale clinical trial of an immunomodulatory biologic in combination with a standardized lifestyle intervention.

ERAPSO: Revealing the High Burden of Obesity in German Psoriasis Patients.

INTRODUCTION: Plaque psoriasis is a chronic, systemic-inflammatory disease characterized by skin erythema, plaques and scaling, and is associated with different comorbidities like psoriatic arthritis, obesity, and cardiometabolic diseases. Obesity aggravates cardiovascular risk in psoriasis patients and can negatively affect psoriasis disease severity with proinflammatory adipocytokine production by adipocytes and infiltrated immune cells. METHODS: An online survey on nutrition and physical activity in psoriasis participants (ERAPSO) collected cross-sectional data about eating behavior, physical activity, and prevalence of obesity and metabolic syndrome components from 9940 psoriasis participants in Germany. RESULTS: ERAPSO revealed a high burden of obesity in German psoriasis participants with 66.9% overweight or obese (body mass index [BMI] ≥ 25 kg/m2), compared to approximately 50% of the German general population. Affected body surface area (BSA), cardiovascular risk factors, and cardiovascular event frequency increased with increasing BMI. Severe psoriasis was more frequent in overweight participants and impaired engagement in weight loss diets and physical activity. Most German psoriasis participants (90.2%) with BMI ≥ 25 kg/m2 perceived themselves as overweight. A minority (21.2%) were currently exercising with the aim of losing weight, and 12.6% were currently on a weight loss diet. In overweight participants, just 13.3% stated that their physicians and/or health insurance offered specific weight loss programs. CONCLUSION: ERAPSO revealed inadequate medical care of obese psoriasis participants with insufficient support for weight loss through diet or increased physical activity. Although psoriasis participants showed an intact self-perception of obesity, they seemed to lack intrinsic motivation to lose weight, highlighting the need for external support in losing weight via tailored programs. Since psoriasis severity correlates with impairment in diets and sports, treating psoriasis adequately may allow participants to follow weight loss programs more successfully. FUNDING: Novartis Pharma GmbH, Nuremberg, DE.

Impact of Secukinumab on Endothelial Dysfunction and Other Cardiovascular Disease Parameters in Psoriasis Patients over 52 Weeks.

Psoriasis increases the risk of cardiovascular (CV) disease. Secukinumab, a fully human monoclonal antibody against IL-17A, shows significant efficacy in psoriasis, but effects on CV markers are unknown. CARIMA (Evaluation of Cardiovascular Risk Markers in Psoriasis Patients Treated with Secukinumab) was a 52-week, randomized, double-blind, placebo-controlled, exploratory trial in patients with moderate to severe plaque psoriasis without clinical CV disease. Patients were randomly assigned to receive 300 mg or 150 mg secukinumab until week 52 or to receive placebo until week 12 and then 300 mg or 150 mg secukinumab until week 52. The primary outcome was endothelial function measured by flow-mediated dilation (FMD). Baseline FMD was significantly lower in psoriasis patients than healthy volunteers (4.4 ± 3.9% vs. 6.1 ± 3.3%, P = 0.01). At week 12, baseline-adjusted mean FMD was numerically higher in patients receiving secukinumab versus those receiving placebo, but this difference (300-mg group, +1.2%; 150-mg group, +0.76%; P = 0.223 and P = 0.403 by analysis of covariance) did not reach significance. At week 52, FMD increased across groups. FMD was significantly higher than baseline in patients receiving the label dose of 300 mg secukinumab for 52 weeks (+2.1%, 95% confidence interval = 0.8-3.3; P = 0.0022). Other relevant CV markers were unchanged. CARIMA indicates that secukinumab might have a beneficial effect on CV risk by improving the endothelial function of patients with plaque psoriasis.

Pharmacokinetics of Certoparin During In Vitro and In Vivo Dialysis.

The efficacy and safety of certoparin in the prophylaxis of clotting during hemodialysis have recently been proven. Different to other low-molecular weight heparins (LMWHs), certoparin does not accumulate in maintenance dialysis patients for unknown reasons. The purpose of the present study was to examine the impact of the dialysis procedure on the removal of certoparin. In a subgroup of the MEMBRANE study consisting of 12 patients, the pharmacokinetics of certoparin during hemodialysis was determined by means of the anti-Xa activity. In addition, the elimination of certoparin into continuously collected dialysate was assessed. Further, in vitro experiments with human blood-simulating high-flux hemodialysis and hemofiltration were performed to quantify the elimination and the sieving coefficients SK of the two LMWHs certoparin and enoxaparin compared with unfractionated heparin (UFH). The surrogate marker middle molecules inulin and myoglobin served as reference solutes during the experiments. Finally, the adsorption of (125) iodine-radiolabeled certoparin onto the synthetic dialysis membrane was quantified. The clinical study reconfirmed the absence of bioaccumulation of certoparin with anti-Xa activities between <0.01 and 0.02 IU/mL after 24 h. A short plasma half-life time of 2.0 ± 0.7 h was determined during hemodialysis. Of the total certoparin dose injected intravenously prior to hemodialysis, only 2.7% was eliminated into dialysate. The in vitro experiments further revealed only 6% of certoparin to be adsorbed onto the dialysis membrane. The anti-Xa activities of certoparin and enoxaparin slowly declined during in vitro hemofiltration to 87.3 ± 5.5 and 82.5 ± 9.4% of baseline, respectively, while inulin and myoglobin concentrations rapidly decreased. The anti-Xa activity of UFH remained unchanged. The SK of both LMWH and UFH was very low in hemofiltration and particularly in hemodialysis with values ≤0.1. The elimination kinetics during hemodialysis suggests strong protein-binding of certoparin. Different from LMWH significantly cleared by the kidneys, the relatively short half-life time of certoparin of only 2 h during hemodialysis allows a more reliable control of the anti-coagulatory effects and decreases the risk of bleeding complications. Dialytic removal does not significantly contribute to the clearance of certoparin in maintenance dialysis patients.

Effects of 8,000 IU aXa long-term prophylaxis with certoparin on the incidence of hyperkalemia in patients with coronary heart disease--a post-hoc analysis of the PARAT trial.

BACKGROUND: Hyperkalemia is an infrequent but potentially serious complication of low molecular weight heparin (LMWH) use. While there are a number of trials comparing LMWH to unfractionated heparin (UFH) there is no comparison of the risk with LMWH versus placebo. Aim of the present post-hoc analysis of the PARAT trial was the description of serum potassium levels with certoparin compared to placebo. RESULTS: PARAT was a double-blind, placebo-controlled, randomized trial in patients with coronary artery disease receiving either 8,000 I.U. aXa per day or placebo. Serum potassium was monitored at baseline and at scheduled follow-up visits at 2 and 4-6 weeks and 3 and 4-6 months. Statistical evaluation included paired, two sided t-test for each of the treatment groups to compare baseline and follow-up values. A total of 117 patients (59 certoparin, 58 placebo) were included with a mean age of 59 years and 84.6% male gender. There was a statistically significant increase in serum potassium at two weeks after discharge compared to baseline (p<0.001) in either group which remained elevated throughout the three months treatment phase. Differences between treatment groups were not statistically significant. After treatment discontinuation at the three months' visit serum potassium returned to normal values (p=n.s. vs. baseline) in both groups. Overall 12 out of 59 patients receiving certoparin (20.3%) and 11 out of 58 patients receiving placebo (19.0%) experienced hyperkalemia based on threshold of >5.0 mmol/l at any time during the observation. CONCLUSIONS: We conclude that there is no incremental risk of hyperkalemia with certoparin up to 8,000 I.U. aXa per day versus placebo in patients with coronary artery disease. The increase in serum potassium values in either group calls for clinical surveillance and the consideration of further risk factors predisposing to hyperkalemia.

Treatment of deep vein thrombosis in patients with pulmonary embolism: subgroup analysis on the efficacy and safety of certoparin vs. unfractionated heparin.

The objective of this subgroup analysis of the pivotal studies NMH-TH-3 and NMH-TH-4 was to investigate the incidence of recurrent events of venous thromboembolism in patients with acute deep vein thrombosis (DVT) with and without pulmonary embolism treated with subcutaneous fixed-dose low-molecular-weight heparin certoparin or intravenous unfractionated heparin (UFH). To assess whether the efficacy of the two treatments is modified by the presence or absence of pulmonary embolism, a P value for subgroup by treatment interaction was calculated using logistic regression. The rate of recurrent venous thromboembolic events, defined as DVT, pulmonary embolism and death due to pulmonary embolism, was observed over 6 months. After 6 months of follow-up, 6.58% of patients with pulmonary embolism at baseline treated with certoparin (5/76) compared with 11.5% of patients with pulmonary embolism at baseline treated with UFH (7/61) had a venous thromboembolic event [relative risk (RR) = 0.57, confidence interval (CI) = 0.19-1.72]. In the group of patients without pulmonary embolism at baseline, 2.82% of patients treated with certoparin (23/816) and 4.63% of patients treated with UFH (37/800) had a venous thromboembolic event (RR = 0.61, CI = 0.37-1.02). The test for interaction between the groups of patients with and without pulmonary embolism was not significant (P = 0.886). The same was true for the safety results with regard to major bleedings and death. These data suggest that the recommendation for the use of certoparin in the treatment of isolated DVT can safely be extended to treatment of DVT in patients concomitantly suffering from pulmonary embolism.

Prospective, non-interventional study on the real-world use of certoparin in daily practice--the PROMEMBER study.

BACKGROUND: We aimed to verify safety and effectiveness of certoparin real-world use and to identify predictors of thromboembolic events or bleeding. METHODS: This was a non-interventional study documenting patients at hospital- or office-based physicians. RESULTS: Patients' (n = 1407) mean age was 53.7 ± 16.1 years. Reason for certoparin use was prophylaxis of venous thromboembolism in 1331 (94.6%) patients and treatment in 76 (5.4%) patients. In only 11.4% of those receiving prophylaxis and 13.2% of those receiving treatment dosing, duration and schedule of certoparin were within label. There were 2 patients with deep venous thrombosis ([DVT], no pulmonary embolism [PE]), and 51 patients (3.8%) with minor (nonmajor) bleeding complications in patients receiving prophylaxis. Two patients treated with certoparin had recurrent DVT and one had PE (one patient with minor bleeding). CONCLUSIONS: Certoparin is very effective in real world. This also applies to patients in whom clinical decision making leads to an alteration of recommended application.

A multi-center, prospective, open-label, 8-week study of certoparin for anticoagulation during maintenance hemodialysis--the membrane study.

BACKGROUND: Adequate anticoagulation is prerequisite for effective hemodialysis to prevent clotting in the extracorporeal circuit. We aimed providing first data on the efficacy and safety of the low-molecular-weight heparin certoparin in this setting. METHODS: Multicenter, open-label, 8-week trial. Patients received a single dose of 3,000 IU certoparin i.v. with additional titration steps of 600 IU and/or continuous infusion if necessary. RESULTS: 120 patients were screened, 109 enrolled (median age 71; range 26-90 years) and 106 available for efficacy analyses. The percentage of unsatisfactory dialysis results at 8 weeks due to clotting or bleeding, was 1.9% (n = 2/106; 95% confidence interval [CI] 0.23-6.65%); no major bleeding. 1.9% had moderate/severe clotting in the lines/bubble catcher and 2.8% in the dialyser at week 8. 15.7 ± 14.3% of the dialysis filters' visual surface area was showing redness. In subgroups of patients receiving median doses of 3000 ± 0, 3000 (2400-6000) and 4200 (3000-6600) IU, plasma aXa levels at baseline, 4 and 8 weeks were 0.24 [95%CI 0.21-0.27], 0.33 [0.27-0.40] and 0.38 [0.33-0.45] aXa IU/ml at 2 h. C48h was 0.01 [0.01-0.02] aXa IU at all visits. At baseline and 4 weeks AUC0-48h was 2.66 [2.19-3.24] and 3.66 [3.00-4.45] aXa IU*h/ml. In 3.0% of dialyses (n = 83/2724) prolonged fistula compression times were documented. Eight patients (7.34%) had at least one episode of minor bleeding. 4) 85.3% of patients had any adverse event, 9.2% were serious without suspected drug relation; and in 32 patients a drug-relation was suspected. CONCLUSIONS: Certoparin appears effective and safe for anticoagulation in patients undergoing maintenance hemodialysis.

Comparison of 3,000 and 5,000 IU aXa/day certoparin in the prevention of deep-vein thrombosis after total hip replacement.

BACKGROUND: The aim was to investigate, whether 5,000 IUaXa/day certoparin lowers the incidence of deep vein thrombosis (DVT) in patients undergoing elective hip replacement surgery vs. 3,000 IUaXa/day. Double-blind, multicenter, randomised trial in 500 patients. Primary endpoint: incidence of symptomatic or asymptomatic DVT (bilateral ascending venography). RESULTS: Mean age was 71 ± 10 years with a higher prevalence of previous DVT (8vs.4%) and pulmonary embolism (PE) (4vs.1%) in the high dose group. Mean duration of surgery was 82 ± 32 and 85 ± 36 min. DVT was detected in 28 (11.1%) of the low dose and 35 (14.1%) of the high dose group (p = n.s.). Combined distal-proximal DVT was observed in 5 (2%) and 4 (1.6%) patients respectively. No difference in bleeding events was found. CONCLUSION: This trial confirms prior data showing that the conventional dosage of 3,000 IU aXa is effective and safe for the prevention of venous thromboembolic events after hip replacement surgery.

Bridging with the Low molecular weight heparin certoparin in patients requiring temporary discontinuation of oral anticoagulation - the non-interventional, retrospective REMEMBER study.

BACKGROUND: Bridging of a temporal discontinuation of oral anticoagulation with low molecular weight heparins (LMWH) is common practice. Specific data on the LMWH certoparin are however limited despite its wide spread use. We aimed to assess effectiveness and safety of certoparin in this indication under real world conditions. METHODS: Retrospective, non-controlled, non-interventional study at hospital- and office based physicians in Germany documenting 259 patients undergoing surgery (trauma, orthopaedic or general), endoscopy or a coronary intervention and who were receiving certoparin to bridge the temporal discontinuation of oral anticoagulation. RESULTS: Patients had a mean age of 70.4 ± 11.5 years, a body weight of 81.9 ± 16.2 kg and 40.9% were female. Most received oral anticoagulation because of atrial fibrillation (57.5%) and most underwent surgery (55.6%). Oral anticoagulant treatment was discontinued a mean of 6.4 ± 2.3 days prior to the intervention and resumed 3.6 ± 3.8 days after the intervention. Certoparin, used in 74.9% of patients at a full therapeutic dose of 1 x 8,000 IE, was started about 3 days after VKA discontinuation when the mean INR was 1.7 ± 0.4 and patients received the last dose of certoparin a mean of 16.2 ± 7.1 h before the intervention. It was resumed 12.2 ± 7.7 h after the intervention and stopped at day 7 when the INR had reached 2.2 ± 0.4. No case of clinically overt deep vein thrombosis and no case of pulmonary embolism was reported. Seven patients (2.7%) had any minor and 4 (1.5%) any major bleeding complication. CONCLUSIONS: The analysis suggests that certoparin is safe and effective to bridge the temporal discontinuation of oral anticoagulation during surgery, endoscopy or a coronary intervention. It further confirms that current use patterns are appropriate in daily practice in Germany to ensure patient safety.

Low-molecular-weight heparin versus placebo for the prevention of venous thromboembolism in metastatic breast cancer or stage III/IV lung cancer.

In 2 double-blind studies, ambulatory patients with objectively proven, disseminated metastatic breast carcinoma (TOPIC-1) or stage III/IV non-small-cell lung carcinoma (TOPIC-2) were randomized to certoparin 3000 IU or placebo subcutaneously once daily, for 6 months. Primary efficacy outcome was objectively confirmed symptomatic or asymptomatic venous thromboembolism (VTE). Safety outcomes included bleeding (major and minor), and thrombocytopenia. TOPIC-1 was halted after an interim analysis. Venous thromboembolism occurrence was not different between treatment groups in TOPIC-1 (4% treated with certoparin, 7 of 174 vs 4% receiving placebo, 7 of 177, odds ratio [OR] 1.02; 95% confidence interval [CI] 0.30-3.48) and in TOPIC-2 (4.5%, 12 of 268) vs 8.3%, 22 of 264, respectively, OR 0.52; CI 0.23-1.12). Mortality was not different between groups. A post hoc analysis showed certoparin significantly reduced VTE in stage IV lung carcinoma (3.5% vs 10.2%; P = .032) without increased bleeding. In conclusion, thrombosis risk and prophylactic benefit was highest in stage IV lung carcinoma patients.

Heparin based prophylaxis to prevent venous thromboembolic events and death in patients with cancer - a subgroup analysis of CERTIFY.

BACKGROUND: Patients with cancer have an increased risk of VTE. We compared VTE rates and bleeding complications in 1) cancer patients receiving LMWH or UFH and 2) patients with or without cancer. METHODS: Acutely-ill, non-surgical patients ≥ 70 years with (n = 274) or without cancer (n = 2,965) received certoparin 3,000 UaXa o.d. or UFH 5,000 IU t.i.d. for 8-20 days. RESULTS: 1) Thromboembolic events in cancer patients (proximal DVT, symptomatic non-fatal PE and VTE-related death) occurred at 4.50% with certoparin and 6.03% with UFH (OR 0.73; 95% CI 0.23-2.39). Major bleeding was comparable and minor bleedings (0.75 vs. 5.67%) were nominally less frequent. 7.5% of certoparin and 12.8% of UFH treated patients experienced serious adverse events. 2) Thromboembolic event rates were comparable in patients with or without cancer (5.29 vs. 4.13%) as were bleeding complications. All cause death was increased in cancer (OR 2.68; 95%CI 1.22-5.86). 10.2% of patients with and 5.81% of those without cancer experienced serious adverse events (OR 1.85; 95% CI 1.21-2.81). CONCLUSIONS: Certoparin 3,000 UaXa o.d. and 5,000 IU UFH t.i.d. were equally effective and safe with respect to bleeding complications in patients with cancer. There were no statistically significant differences in the risk of thromboembolic events in patients with or without cancer receiving adequate anticoagulation. TRIAL REGISTRATION: clinicaltrials.gov, NCT00451412.

Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting.

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated 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 (GlyRalpha1-4 and -beta). 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 GlyRalpha1 and -alpha3 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 beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, 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 alpha3 and alpha4 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.

Mapping of disulfide bonds within the amino-terminal extracellular domain of the inhibitory glycine receptor.

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

Recessive hyperekplexia mutations of the glycine receptor alpha1 subunit affect cell surface integration and stability.

The human neurological disorder hyperekplexia is frequently caused by recessive and dominant mutations of the glycine receptor alpha1 subunit gene, GLRA1. Dominant forms are mostly attributed to amino acid substitutions within the ion pore or adjacent loops, resulting in altered channel properties. Here, the biogenesis of glycine receptor alpha1 subunit mutants underlying recessive forms of hyperekplexia was analyzed following recombinant expression in HEK293 cells. The alpha1 mutant S231R resulted in a decrease of surface integrated protein, consistent with reduced maximal current values. Decreased maximal currents shown for the recessive alpha1 mutant I244N were associated with protein instability, rather than decreased surface integration. The recessive mutants R252H and R392H encode exchanges of arginine residues delineating the intracellular faces of transmembrane domains. After expression, the mutant R252H was virtually absent from the cell surface, consistent with non-functionality and the importance of the positive charge for membrane integration. Surface expression of R392H was highly reduced, resulting in residual chloride conductance. Independent of the site of the mutation within the alpha1 polypeptide, metabolic radiolabelling and pulse chase studies revealed a shorter half-life of the full-length alpha1 protein for all recessive mutants as compared to the wild-type. Treatment with the proteasome blocker, lactacystin, significantly increased the accumulation of alpha1 mutants in intracellular membranes. These observations indicated that the recessive alpha1 mutants are recognized by the endoplasmatic reticulum control system, and degraded via the proteasome pathway. Thus, the lack of glycinergic inhibition associated with recessive hyperekplexia may be attributed to sequestration of mutant subunits within the endoplasmatic reticulum quality control system.

Novel regulatory site within the TM3-4 loop of human recombinant alpha3 glycine receptors determines channel gating and domain structure.

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants alpha3L and alpha3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3-4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3-4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3-4 loops of alpha3L and alpha3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of alpha3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3-4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3-4 loop of alpha3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.