The serological diversity of serum IgG/IgA/IgM against SARS-CoV-2 nucleoprotein, spike, and receptor-binding domain and neutralizing antibodies in patients with COVID-19 in Japan.

Background: We compared the temporal changes of immunoglobulin M (IgM), IgG, and IgA antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (N), spike 1 subunit (S1), and receptor-binding domain (RBD), and neutralizing antibodies (NAbs) against SARS-CoV-2 in patients with coronavirus disease 2019 (COVID-19) to understand the humoral immunity in COVID-19 patients for developing drugs and vaccines for COVID-19. Methods: A total of five confirmed COVID-19 cases in Nissan Tamagawa Hospital in early August 2020 were recruited in this study. Using a fully automated chemiluminescence immunoassay analyzer, we measured the levels of IgG, IgA, and IgM against SARS-CoV-2 N, S1, and RBD and NAbs against SARS-CoV-2 in COVID-19 patients' sera acquired multiple times in individuals from 0 to 76 days after symptom onset. Results: IgG levels against SARS-CoV-2 structural proteins increased over time in all cases but IgM and IgA levels against SARS-CoV-2 showed different increasing trends among individuals in the early stage. In particular, we observed IgA increasing before IgG and IgM in some cases. The NAb levels were more than cut-off value in 4/5 COVID-19 patients some of whose antibodies against RBD did not exceed the cut-off value in the early stage. Furthermore, NAb levels against SARS-CoV-2 increased and kept above cut-off value more than around 70 days after symptom onset in all cases. Conclusion: Our findings indicate COVID-19 patients should be examined for IgG, IgA, and IgM against SARS-CoV-2 structural proteins and NAbs against SARS-CoV-2 to analyze the diversity of patients' immune mechanisms.

Measurement of SARS-CoV-2 Antibody Titers Improves the Prediction Accuracy of COVID-19 Maximum Severity by Machine Learning in Non-Vaccinated Patients.

Numerous studies have suggested that the titers of antibodies against SARS-CoV-2 are associated with the COVID-19 severity, however, the types of antibodies associated with the disease maximum severity and the timing at which the associations are best observed, especially within one week after symptom onset, remain controversial. We attempted to elucidate the antibody responses against SARS-CoV-2 that are associated with the maximum severity of COVID-19 in the early phase of the disease, and to investigate whether antibody testing might contribute to prediction of the disease maximum severity in COVID-19 patients. We classified the patients into four groups according to the disease maximum severity (severity group 1 (did not require oxygen supplementation), severity group 2a (required oxygen supplementation at low flow rates), severity group 2b (required oxygen supplementation at relatively high flow rates), and severity group 3 (required mechanical ventilatory support)), and serially measured the titers of IgM, IgG, and IgA against the nucleocapsid protein, spike protein, and receptor-binding domain of SARS-CoV-2 until day 12 after symptom onset. The titers of all the measured antibody responses were higher in severity group 2b and 3, especially severity group 2b, as early as at one week after symptom onset. Addition of data obtained from antibody testing improved the ability of analysis models constructed using a machine learning technique to distinguish severity group 2b and 3 from severity group 1 and 2a. These models constructed with non-vaccinated COVID-19 patients could not be applied to the cases of breakthrough infections. These results suggest that antibody testing might help physicians identify non-vaccinated COVID-19 patients who are likely to require admission to an intensive care unit.

Serologic Survey of IgG Against SARS-CoV-2 Among Hospital Visitors Without a History of SARS-CoV-2 Infection in Tokyo, 2020-2021.

BACKGROUND: Tokyo, the capital of Japan, is a densely populated city of >13 million people, so the population is at high risk of epidemic severe acute respiratory coronavirus 2 (SARS-CoV-2) infection. A serologic survey of anti-SARS-CoV-2 IgG would provide valuable data for assessing the city's SARS-CoV-2 infection status. Therefore, this cross-sectional study estimated the anti-SARS-CoV-2 IgG seroprevalence in Tokyo. METHODS: Leftover serum of 23,234 hospital visitors was tested for antibodies against SARS-CoV-2 using an iFlash 3000 chemiluminescence immunoassay analyzer (Shenzhen YHLO Biotech, Shenzhen, China) with an iFlash-SARS-CoV-2 IgG kit (YHLO) and iFlash-SARS-CoV-2 IgG-S1 kit (YHLO). Serum samples with a positive result (≥10 AU/mL) in either of these assays were considered seropositive for anti-SARS-CoV-2 IgG. Participants were randomly selected from patients visiting 14 Tokyo hospitals between September 1, 2020 and March 31, 2021. No participants were diagnosed with coronavirus disease 2019 (COVID-19), and none exhibited COVID-19-related symptoms at the time of blood collection. RESULTS: The overall anti-SARS-CoV-2 IgG seroprevalence among all participants was 1.83% (95% confidence interval [CI], 1.66-2.01%). The seroprevalence in March 2021, the most recent month of this study, was 2.70% (95% CI, 2.16-3.34%). After adjusting for population age, sex, and region, the estimated seroprevalence in Tokyo was 3.40%, indicating that 470,778 individuals had a history of SARS-CoV-2 infection. CONCLUSIONS: The estimated number of individuals in Tokyo with a history of SARS-CoV-2 infection was 3.9-fold higher than the number of confirmed cases. Our study enhances understanding of the SARS-CoV-2 epidemic in Tokyo.

Response kinetics of different classes of antibodies to SARS-CoV2 infection in the Japanese population: The IgA and IgG titers increased earlier than the IgM titers.

To better understand the immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with COVID-19, it is important to investigate the kinetics of the antibody responses and their associations with the clinical course in different populations, since there seem to be considerable differences between Western and Asian populations in the clinical features and spread of COVID-19. In this study, we serially measured the serum titers of IgM, IgG and IgA antibodies generated against the nucleocapsid protein (NCP), S1 subunit of the spike protein (S1), and receptor-binding domain in the S1 subunit (RBD) of SARS-CoV-2 in Japanese individuals with COVID-19. Among the IgM, IgG, and IgA antibodies, IgA antibodies against all of the aforementioned viral proteins were the first to appear after the infection, and IgG and/or IgA seroconversion often preceded IgM seroconversion. In regard to the timeline of the antibody responses to the different viral proteins (NCP, S1 and RBD), IgA against NCP appeared than IgA against S1 or RBD, while IgM and IgG against S1 appeared earlier than IgM/IgG against NCP or RBD. The IgG responses to all three viral proteins and responses of all three antibody classes to S1 and RBD were sustained for longer durations than the IgA/IgM responses to all three viral proteins and responses of all three antibody classes to NCP, respectively. The seroconversion of IgA against NCP occurred later and less frequently in patients with mild COVID-19. These results suggest possible differences in the antibody responses to SARS-CoV-2 antigens between the Japanese and Western populations.

Association of the Serum Levels of the Nucleocapsid Antigen of SARS-CoV-2 With the Diagnosis, Disease Severity, and Antibody Titers in Patients With COVID-19: A Retrospective Cross-Sectional Study.

Background: Several types of laboratory tests for COVID-19 have been established to date; however, the clinical significance of the serum SARS-CoV-2 nucleocapsid (N) antigen levels remains to be fully elucidated. In the present study, we attempted to elucidate the usefulness and clinical significance of the serum N antigen levels. Methods: We measured the serum N antigen levels in 391 serum samples collected from symptomatic patients with a confirmed diagnosis of COVID-19 and 96 serum samples collected from patients with non-COVID-19, using a fully automated chemiluminescence immunoassay analyzer. Results: Receiver operating characteristic analysis identified the optimal cutoff value of the serum N antigen level (cutoff index, based on Youden's index) as 0.255, which yielded a sensitivity and specificity for the diagnosis of COVID-19 of 91.0 and 81.3%, respectively. The serum N antigen levels were significantly higher in the patient groups with moderate and severe COVID-19 than with mild disease. Moreover, a significant negative correlation was observed between the serum N antigen levels and the SARS-CoV-2 IgG antibody titers, especially in patients with severe COVID-19. Conclusion: Serum N antigen testing might be useful both for the diagnosis of COVID-19 and for obtaining a better understanding of the clinical features of the disease.

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan.

PCR methods are presently the standard for the diagnosis of Coronavirus disease 2019 (COVID-19), but additional methodologies are needed to complement PCR methods, which have some limitations. Here, we validated and investigated the usefulness of measuring serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the iFlash3000 CLIA analyzer. We measured IgM and IgG titers against SARS-CoV-2 in sera collected from 26 PCR-positive COVID-19 patients, 53 COVID-19-suspected but PCR-negative patients, and 20 and 100 randomly selected non-COVID-19 patients who visited our hospital in 2020 and 2017, respectively. The repeatability and within-laboratory precision were obviously good in validations, following to the CLSI document EP15-A3. Linearity was also considered good between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG, while the linearity curves plateaued above the upper measurement range. We also confirmed that the seroconversion and no-antibody titers were over the cutoff values in all 100 serum samples collected in 2017. These results indicate that this measurement system successfully detects SARS-CoV-2 IgM/IgG. We observed four false-positive cases in the IgM assay and no false-positive cases in the IgG assay when 111 serum samples known to contain autoantibodies were evaluated. The concordance rates of the antibody test with the PCR test were 98.1% for SARS-CoV-2 IgM and 100% for IgG among PCR-negative cases and 30.8% for SARS-CoV-2 IgM and 73.1% for SARS-CoV-2 IgG among PCR-positive cases. In conclusion, the performance of this new automated method for detecting antibody against both N and S proteins of SARS-CoV-2 is sufficient for use in laboratory testing.

Time course of the sensitivity and specificity of anti-SARS-CoV-2 IgM and IgG antibodies for symptomatic COVID-19 in Japan.

The accurate and prompt diagnosis of SARS-CoV-2 infection is required for the control and treatment of the coronavirus infection disease 2019 (COVID-19). In this study, we aimed to investigate the time courses of the anti-severe acute corona respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM and IgG titers and to evaluate the sensitivity and specificity of such tests according to the specific day after the onset of COVID-19 among a patient population in Japan. We measured the titers of SARS-CoV-2 IgM and IgG in sera from 105 subjects, including 26 symptomatic COVID-19 patients, using chemiluminescent immunoassay (CLIA) methods utilizing magnetic beads coated with SARS-CoV-2 nucleocapsid protein and spike protein. The results of a ROC analysis suggested the possibility that the cutoff values in Japan might be lower than the manufacturer's reported cutoff (10 AU/mL): 1  AU/mL for IgM and 5  AU/mL for IgG. The sensitivity of the test before Day 8 after symptom onset was less than 50%; at Days 9-10, however, we obtained a much higher sensitivity of 81.8% for both IgM and IgG. At 15 days or later after symptom onset, the SARS-CoV-2 IgG test had a sensitivity of 100%. These results suggest that if the number of days since disease onset is taken into consideration, these antibody tests could be very useful for the diagnosis of COVID-19 and similar diseases.

Efficacy of Chemiluminescence Immunoassays on VCA-IgA and EBNA1-IgA Antibodies of Epstein-Barr Virus in Diagnosing Nasopharyngeal Carcinoma.

Background: IgA antibodies against Epstein-Barr virus (EBV) capsid antigen (VCA) and nuclear antigen 1 (EBNA1) have been proposed to facilitate the diagnosis and early detection of nasopharyngeal carcinoma (NPC) in high-incidence regions. However, while new methodologies and new platforms for the detection of VCA-IgA and EBNA1-IgA have become available, proper interassay simultaneous comparisons have not been carried out. The study was to compare the performance of the chemiluminescent immunoassays (CLIA) and enzyme-linked immunosorbent assay (ELISA) for VCA-IgA and EBNA1-IgA antibodies, and to evaluate the levels of EBV antibodies in healthy population from different areas of China. Methods: CLIA and ELISA for VCA-IgA and EBNA1-IgA were performed in NPC and healthy populations from high-incidence areas of NPC in South China (N=555), medium-incidence areas of NPC in Central China (N=318) and low-incidence areas of NPC in North China (N=379), and the results were compared and analyzed. Results: (1) The highest sensitivity in total, early and advanced NPC were 91.5% (CLIA for VCA-IgA), 86.4% (CLIA and ELISA-2 for EBNA1-IgA) and 93.6% (CLIA for VCA-IgA). However, the specificity of EBV-IgA measured by CLIA was relatively lower than ELISA. The top three seromarkers with the largest AUC was CLIA for VCA-IgA (AUC: 0.929, 95% CI: 0.905-0.953), ELISA-2 for EBNA1-IgA (AUC: 0.922, 95% CI: 0.896-0.947) and CLIA for EBNA1-IgA (AUC:0.919, 95% CI: 0.893-0.945), respectively. The positive and negative coincidence rates of the two EBNA1-IgA kits were 69.5% and 91.9%, respectively. However, the coincidence rates of VCA-IgA were relatively low. CLIA kits had good repeatability between different laboratories. (2) The positive rates of EBV-IgA antibodies were relatively high in high-incidence areas of NPC (P < 0.017), while there was no significant difference in the antibody positive rates between medium-incidence areas and low-incidence areas of NPC (P > 0.05). Conclusions: The performance of EBV-IgA antibodies measured by CLIA has good repeatability, higher sensitivity and similar specificity. The higher EBV-IgA positive rate in healthy subjects by CLIA raises concern about its suitability for NPC-risk screening and requires further analysis.

Development and multicenter performance evaluation of fully automated SARS-CoV-2 IgM and IgG immunoassays.

Objectives: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread globally. The laboratory diagnosis of SARS-CoV-2 infection has relied on nucleic acid testing; however, it has some limitations, such as low throughput and high rates of false negatives. Tests of higher sensitivity are needed to effectively identify infected patients. Methods: This study has developed fully automated chemiluminescent immunoassays to determine IgM and IgG antibodies to SARS-CoV-2 in human serum. The assay performance has been evaluated at 10 hospitals. Clinical specificity was evaluated by measuring 972 hospitalized patients and 586 donors of a normal population. Clinical sensitivity was assessed on 513 confirmed cases of SARS-CoV-2 by RT-PCR. Results: The assays demonstrated satisfied assay precision with coefficient of variation of less than 4.45%. Inactivation of specimen did not affect assay measurement. SARS-CoV-2 IgM showed clinical specificity of 97.33 and 99.49% for hospitalized patients and the normal population respectively, and SARS-CoV-2 IgG showed clinical specificity of 97.43 and 99.15% respectively. SARS-CoV-2 IgM showed clinical sensitivity of 82.54, 92.93, and 84.62% before 7 days, 7-14 days, and after 14 days respectively, since onset of symptoms, and SARS-CoV-2 IgG showed clinical sensitivity of 80.95, 97.98, and 99.15% respectively at the same time points above. Conclusions: We have developed fully automated immunoassays for detecting SARS-CoV-2 IgM and IgG antibodies in human serum. The assays demonstrated high clinical specificity and sensitivity, and add great value to nucleic acid testing in fighting against the global pandemic of the SARS-CoV-2 infection.

Cyclic and safety utilisation of Cu polluted biogas residue in saline-alkali soil.

The proper disposal of copper (Cu) polluted plant residues after phytoremediation has attracted extensive attention. In this study, the Cu-polluted biogas residue produced through anaerobic digestion was applied directly. Wheat, soybean and pakchoi were grown in pots for four seasons over two years. The application dosage of Cu-polluted biogas residue was evaluated by measuring growth conditions of crops, Cu content in edible parts, and amelioration of saline-alkali soil. The results showed that the biomass of the crops, the content of soil organic matter, total N and available P and microbial diversity can be improved, and the Cu concentration of the edible parts was all lower than limit standard. Amendment with 2% biogas residue enhanced the growth of beneficial bacteria and fungi, and decreased the relative abundances of potentially pathogenic fungi in the saline-alkali soil. The results of this study provide a basis for the safe utilisation of copper-polluted plant residues.

Performance evaluation of a new high throughput Mindray BS-2000M1 clinical chemistry system.

OBJECTIVE: To evaluate the photometry technical and analytical performance of a newly launched Mindray BS-2000M1 clinical chemistry system (BS-2000M1). DESIGN AND METHODS: The photometric parameters were evaluated according to the China Food and Drug Administration (CFDA) Automatic Chemistry Analyzer Guideline. The precision, accuracy, linearity and interference were evaluated according to CLSI protocols EP5-A2, EP9-A2, EP6-A and EP7-A2 respectively. The trueness verification on Ca(2+), Mg(2+), P(-) and Cl(-) was conducted by comparing with the reference methods using fresh samples. RESULTS: The photometer accuracy, precision, linearity, stability and stray light at 340 nm were acceptable. The within-run coefficients of variation (CVs) ranged from 0.16% to 2.13% and the total CV ranged from 0.64% to 4.12%. A good correlation (R>0.95) of method comparison between BS-2000M1 and our reference system was observed for most of the parameters tested with exception of Ca(2+) (R=0.85), Mg(2+) (R=0.71), P(-) (R=0.96, Slope=0.88), Cl(-) (R=0.93), and ASO (R=0.94, Slope=0.93, intercept=-8.81). The trueness verification on Ca(2+), Mg(2+), P(-) and Cl(-) showed acceptable results on both BS-2000M1 and our reference systems. Linearity study showed acceptable linearity range for all parameters. Significant interferences occurred for some evaluated parameters, but were identical to the manufacturer statement. CONCLUSIONS: Mindray BS-2000M1 achieved the desirable photometry technical and analytical performance, and is therefore suitable for its utilization in modern clinical laboratories.

Circadian rhythm of circulating fibroblast growth factor 21 is related to diurnal changes in fatty acids in humans.

BACKGROUND: Fibroblast growth factor (FGF) 21 is an endocrine factor actively involved in glucose and lipid metabolism in rodents. However, little is known about its physiological function and regulation in humans. This study investigated the diurnal changes in circulating FGF21 concentrations and their association with other metabolic markers in both obese and lean individuals. METHODS: A total of 36 volunteers were assigned to 2 groups. One group received 3 standardized meals and another group was fasted for 24 h. Blood samples were drawn every 30 min throughout a 24-h period. Circulating FGF21 concentrations were measured with an in-house chemiluminescence immunoassay. The effects of fatty acids on hepatic production of FGF21 were determined by using real-time PCR. RESULTS: In both the fasting and standardized meals groups, circulating FGF21 began to rise at midnight, reaching a peak in the early morning and then declining to basal concentrations early in the afternoon. Baseline concentrations of circulating FGF21 were much higher in obese individuals than in lean individuals (P < 0.05). However, the magnitude of the nocturnal rise in circulating FGF21 was significantly blunted in obese individuals. The 24-h oscillatory pattern of circulating FGF21 resembled that of free fatty acids and cortisol, but was opposite to the patterns of insulin and glucose. Unsaturated fatty acids induced time-dependent expression of FGF21 mRNA in human hepatocytes. CONCLUSIONS: These findings support the role of FGF21 as an important metabolic regulator that integrates the circadian rhythm with energy homeostasis in humans. Diurnal rhythms of circulating FGF21 could be partly caused by the oscillation of free fatty acids.

Inhibition of cholesterol biosynthesis impairs insulin secretion and voltage-gated calcium channel function in pancreatic beta-cells.

Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes.

Targeting of voltage-gated K+ and Ca2+ channels and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins to cholesterol-rich lipid rafts in pancreatic alpha-cells: effects on glucagon stimulus-secretion coupling.

Pancreatic alpha-cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of alpha-cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K(+) (K(V)) and Ca(2+) (Ca(V)) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic alpha-cells and examined their targeting to specialized cholesterol-rich lipid rafts. In alpha-cells, we detected the expression of K(V)4.1/4.3 (A-type current), K(V)3.2/3.3 (delayed rectifier current), Ca(V)1.2 (L-type current), Ca(V)2.2 (N-type current), and the SNARE (synaptosomal-associated protein of 25 kDa, syntaxin 1A, and vesicle-associated membrane protein 2) and SNARE-associated proteins (Munc-13-1 and Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins (syntaxin 1A, synaptosomal-associated protein of 25 kDa, and vesicle-associated membrane protein 2) target to lipid raft domains on alpha-cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin decreased the association of K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins with lipid rafts. This resulted in inhibition of A-type K(V) currents and enhancement of glucagon secretion from alpha-cells. Consistently, capacitance measurements of exocytosis of single alpha-cells showed enhanced exocytosis after membrane cholesterol depletion. Taken together, our results demonstrate the association of K(V)4, Ca(V)1.2, and SNARE proteins with lipid rafts in pancreatic alpha-cells. Glucagon secretion from alpha-cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.

Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25.

Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.

Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels.

We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.

Syntaxin-1A inhibits cardiac KATP channels by its actions on nucleotide binding folds 1 and 2 of sulfonylurea receptor 2A.

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on K(ATP) channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.

Disruption of pancreatic beta-cell lipid rafts modifies Kv2.1 channel gating and insulin exocytosis.

In pancreatic beta-cells, the predominant voltage-gated Ca(2+) channel (Ca(V)1.2) and K(+) channel (K(V)2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K(V)2.1 and Ca(V)1.2, but not K(V)1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on beta-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-beta-cyclodextrin shunts K(V)2.1, Ca(V)1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-beta-cyclodextrin inhibits K(V)2.1 but not Ca(V)1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.

Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways.

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.