Nonselective TRPC channel inhibition and suppression of aminoglycoside-induced premature termination codon readthrough by the small molecule AC1903.

Nonsense mutations, which occur in ∼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4-C1, and TRPC5-C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8-18 µM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake.

Schwarzinicine A inhibits transient receptor potential canonical channels and exhibits overt vasorelaxation effects.

This study investigated the vasorelaxant effects of schwarzinicine A, an alkaloid recently reported from Ficus schwarzii Koord. Regulation of calcium homeostasis in vascular smooth muscle cells (VSMC) is viewed as one of the main mechanisms for controlling blood pressure. L-type voltage-gated calcium channel (VGCC) blockers are commonly used for controlling hypertension. Recently, the transient receptor potential canonical (TRPC) channels were found in blood vessels of different animal species with evidence of their roles in the regulation of vascular contractility. In this study, we studied the mechanism of actions of schwarzinicine A focusing on its regulation of L-type VGCC and TRPC channels. Schwarzinicine A exhibited the highest vasorelaxant effect (123.1%) compared to other calcium channel blockers. It also overtly attenuated calcium-induced contractions of the rat isolated aortae in a calcium-free environment showing its mechanism to inhibit calcium influx. Fluorometric intracellular calcium recordings confirmed its inhibition of hTRPC3-, hTRPC4-, hTRPC5- and hTRPC6-mediated calcium influx into HEK cells with IC50 values of 3, 17, 19 and 7 µM, respectively. The evidence gathered in this study suggests that schwarzinicine A blocks multiple TRPC channels and L-type VGCC to exert a significant vascular relaxation response.

CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

DogCatcher allows loop-friendly protein-protein ligation.

There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.

Potent, selective, and subunit-dependent activation of TRPC5 channels by a xanthine derivative.

BACKGROUND AND PURPOSE: The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium-permeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthine-based TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthine-based activators of these channels. EXPERIMENTAL APPROACH: An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4-C1, TRPC5-C1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+ ]i ) and by patch-clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels. KEY RESULTS: AM237 potently activated TRPC5:C5 channels (EC50 15-20 nM in [Ca2+ ]i assay) and potentiated their activation by sphingosine-1-phosphate but suppressed activation evoked by (-)-englerin A (EA). In patch-clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237-induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4-C1, TRPC5-C1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EA-dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels. CONCLUSIONS AND IMPLICATIONS: This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine-based compounds can activate, potentiate, or inhibit these channels depending on subunit composition.

Remarkable Progress with Small-Molecule Modulation of TRPC1/4/5 Channels: Implications for Understanding the Channels in Health and Disease.

Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na⁺, K⁺ and Ca2+. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes.

The Role of Ubiquitination and Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate in the Degradation of the Adrenomedullin Type I Receptor.

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.

Emergence of multiple cytomegalovirus strains in blood and lung of lung transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) is a major pathogen in lung transplant recipients (LTRs). The emergence of different CMV strains in lung and blood after transplantation has not yet been analyzed. METHODS: In total, 75 serum and 91 broncheoalveolar lavage (BAL) samples obtained from 25 LTRs in the follow-up after transplantation were tested for the presence of different CMV strains. The gB, gN, and gO genes of the CMV isolates were analyzed by subtype-specific PCR, restriction fragment length polymorphism (RFLP), sequencing, and phylogenetic analysis. RESULTS: Mixed CMV-strain populations were detected after cessation of antiviral prophylaxis in up to 80% and 90% of the patients' BAL and serum, respectively, and this was independent of the CMV serostatus of donor and recipient. In five patients, the same single CMV strain was consistently detectable over at least 1 year in lung and blood, although in two of these cases donor and recipient had both been CMV-seropositive. Most CMV strains were distributed in the lung and blood compartment. Symptomatic CMV infection within the first year after transplantation was observed only in patients with mixed CMV-strain populations (P<0.05). CONCLUSION: Most LTRs harbor more than one CMV strain in their lung and blood compartment after cessation of prophylaxis, but the CMV strain distribution within and between the compartments varies between individuals and is not associated with the donor/recipient serostatus. The data further show that compartmentalization of CMV strains in lung versus blood seems to be a rare event and that the presence of mixed CMV-strain infections within the first year after transplantation may be disadvantageous for LTRs.

An unrecognized epidemic of Mycoplasma pneumoniae infection in Vienna.

Mycoplasma pneumoniae infection shows epidemiological peaks with a 2- to 10-fold increased incidence every four to seven years. The regional epidemiology of M. pneumoniae infection is important with regard to empirical antibiotic treatment of community-acquired respiratory tract infections, which are the most common cause for visiting a physician. To date, no data on the epidemiology of M. pneumoniae in central Europe have been published. In the present study, the results of M. pneumoniae serology performed at the Clinical Division of Virology at Vienna General Hospital (a 2,140-bed university teaching hospital with an average of 94,000 admissions/year and 430,000 outpatient visits/year) in the 10-year period from January 1995 to December 2004 were analyzed retrospectively. Antibody titers > or = 1:64 in complement fixation tests were considered indicative of acute or recent mycoplasma infection. The annual total number of serum specimens tested for anti-M. pneumoniae antibodies remained stable throughout the study period (median: 2859 samples/year, range: 2257-3338). The annual median number of patients with high M. pneumoniae titers was 13. A major epidemiological peak (43 patients) was observed in 2000, the epidemic starting in late 1999 and ending in 2001. A surveillance or reporting system for M. pneumoniae infections (i.e. positive serological results for M. pneumoniae) would be useful for physicians caring for patients with community-acquired respiratory tract infections.

Modulation of ion channels by hydrogen sulfide.

SIGNIFICANCE: Evidence of the ability of the gasotransmitter hydrogen sulfide (H(2)S) to serve as a regulator of many physiological functions, including control of blood pressure, regulation of cardiac function, protection of neurons, and cardiomyocytes against apoptosis, and in pain sensation is accumulating. However, the mechanisms accounting for its many actions are not yet well understood. RECENT ADVANCES: Following the pioneering studies of the regulation of N-methyl-d-aspartate receptors and ATP-sensitive K(+) channels by H(2)S, data continue to emerge indicating that H(2)S modulates other ion channel types. This article reviews the numerous, yet diverse, types of ion channels now reported to be regulated by H(2)S. CRITICAL ISSUES: Currently, a critical issue within this field is to determine the mechanisms by which H(2)S regulates ion channels, as well as other target proteins. Mechanisms to account for regulation include direct channel protein sulfhydration, channel redox modulation, effects mediated by interactions with other gasotransmitters (carbon monoxide and nitric oxide), and indirect effects, such as modulation of channel-regulating kinases. Through such modulation of ion channels, novel roles for H(2)S are emerging as important factors in both physiological and pathological processes. FUTURE DIRECTIONS: Increasing current awareness and understanding of the roles and mechanisms of action of ion channel regulation by H(2)S will open opportunities for therapeutic intervention with clear clinical benefits, and inform future therapies. In addition, more sensitive methods for detecting relevant physiological concentrations of H(2)S will allow for clarification of specific ion channel regulation with reference to physiological or pathophysiological settings.

Peroxynitrite mediates disruption of Ca2+ homeostasis by carbon monoxide via Ca2+ ATPase degradation.

AIM: Sublethal carbon monoxide poisoning causes prolonged neurological damage involving oxidative stress. Given the central role of Ca(2+) homeostasis and its vulnerability to stress, we investigated whether CO disrupts neuronal Ca(2+) homeostasis. RESULTS: Cytosolic Ca(2+) transients evoked by muscarine in SH-SY5Y cells were prolonged by CO (applied via the donor CORM-2), and capacitative Ca(2+) entry (CCE) was dramatically enhanced. Ca(2+) store mobilization by cyclopiazonic acid was similarly augmented, as was the subsequent CCE, and that evoked by thapsigargin. Ca(2+) rises evoked by depolarization were also enhanced by CO, and Ca(2+) levels often did not recover in its presence. CO increased intracellular nitric oxide (NO) and all effects of CO were prevented by inhibiting NO formation. However, NO donors did not mimic the effects of CO. The antioxidant ascorbic acid inhibited effects of CO on Ca(2+) signaling, as did the peroxynitrite scavenger, FeTPPS, and CO increased peroxynitrite formation. Finally, CO caused significant loss of plasma membrane Ca(2+)ATPase (PMCA) protein, detected by Western blot, and this was also observed in brain tissue of rats exposed to CO in vivo. INNOVATION: The cellular basis of CO-induced neurotoxicity is currently unknown. Our findings provide the first data to suggest signaling pathways through which CO causes neurological damage, thereby opening up potential targets for therapeutic intervention. CONCLUSION: CO stimulates formation of NO and reactive oxygen species which, via peroxynitrite formation, inhibit Ca(2+) extrusion via PMCA, leading to disruption of Ca(2+) signaling. We propose this contributes to the neurological damage associated with CO toxicity.

Modulation of Ca(2+) signalling in human vascular endothelial cells by hydrogen sulfide.

Hydrogen sulfide (H(2)S) is now recognised as an important endogenous antihypertensive molecule and is synthesised in the vasculature primarily by endothelial cystathionine gamma lyase. Activity of this enzyme, and the production of other vasoactive substances by the endothelium, are subject to modulation by changes of [Ca(2+)](i). Here, we have used microfluorimetry to investigate whether H(2)S can regulate human endothelial [Ca(2+)](i). H(2)S (applied via the donor NaHS, 5-500 microM) caused concentration-dependent rises of [Ca(2+)](i) which were attributable to release from an ATP- and 4-CEP sensitive intracellular pool. Rises of [Ca(2+)](i) evoked by H(2)S were essentially abolished by prior pool depletion. In the absence of external Ca(2+), H(2)S slowed the decay phase of responses to cyclopiazonic acid, but this could not be attributed to the inhibition of Ca(2+) extrusion since the effects of H(2)S were at least additive with the Na(+)/Ca(2+) exchange inhibitors bepridil and SEA 0400 and the Ca(2+) ATPase inhibitor, carboxyeosin. In some but not all the cells, re-exposure to extracellular Ca(2+) following the addition and removal of H(2)S activated capacitative Ca(2+) entry (CCE), and H(2)S increased ATP-evoked (but not thapsigargin-evoked) CCE. Effects of H(2)S were not mediated by energy depletion or production of cyclic ADP ribose. Our data indicate that H(2)S can modulate endothelial [Ca(2+)](i) via multiple mechanisms, and such effects are likely to contribute to this gasotransmitter's beneficial actions.

Hypoxic remodelling of Ca(2+) signalling in proliferating human arterial smooth muscle.

Ca(2+) homeostasis in proliferating smooth muscle (SM) cells strongly influences neointima formation, which can cause failure of coronary artery bypass surgery. During surgical procedures and subsequent revascularization, SM cells are also exposed to a period of hypoxia. Problems with bypass surgery in general involve neointima formation which is in turn dependent on SM proliferation and migration. Here, we have directly monitored [Ca(2+)](i) fluorimetrically in proliferating internal mammary artery (IMA) SM cells, and investigated how this is modulated by chronic hypoxia (CH; 24 h, 2.5% O(2)). IMA is the most successful replacement conduit vessel in bypass grafts. Basal [Ca(2+)](i) was unaffected by CH, but removal of extracellular Ca(2+) evoked far smaller reductions in [Ca(2+)](i) than were seen in normoxic cells. Voltage-gated Ca(2+) entry was suppressed in CH cells, and this was attributable to activation of the transcriptional regulator, hypoxia inducible factor. Furthermore, the relative contributions to voltage-gated Ca(2+) entry of L- and T-type Ca(2+) channels was markedly altered, with T-type channels becoming functionally more important in CH cells. Agonist-evoked mobilization of Ca(2+) from intracellular stores was not affected by CH, whilst subsequent capacitative Ca(2+) entry was modestly suppressed. Our data provide novel observations of the remodelling of Ca(2+) homeostasis by CH in IMASM cells which may contribute to their superior patency as coronary bypass grafts.

Relationship between cytomegalovirus DNA load in epithelial lining fluid and plasma of lung transplant recipients and analysis of coinfection with Epstein-Barr virus and human herpesvirus 6 in the lung compartment.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in lung transplant recipients (LTRs). The aim of the present study was to elucidate the relationship between the CMV DNA load in the lung compartment and that in plasma. For CMV load determination, the level of CMV DNA in plasma and bronchoalveolar lavage (BAL) samples was measured in a total of 97 paired BAL and plasma samples obtained from 25 LTRs. The original virus concentration in the epithelial lining fluid (ELF) was calculated from the BAL samples by correcting for dilution using the urea dilution method. In addition, the load of Epstein-Barr virus (EBV) and that of human herpesvirus 6 (HHV-6) DNA also were determined in BAL samples, recalculated for their concentrations in the ELF, and compared with the CMV DNA load. CMV DNA was found more frequently and at significantly higher levels in the lung compartment than in plasma (P<0.001, Wilcoxon test), and the CMV load in the ELF was associated with symptomatic CMV disease. EBV and HHV-6 were detected in 43.6% and 21.7% of the ELF samples, respectively. A statistically significant association was found between the CMV and EBV DNA loads in the ELF (P<0.001; Spearman's rho=0.651). Thus, in LTRs, determination of the CMV DNA load in the lung compartment may be advantageous compared to monitoring only viremia. The significant relationship between EBV and CMV DNA loads in the ELF of LTRs and its clinical impact require further investigation.

Serum Epstein-Barr virus DNA load in primary Epstein-Barr virus infection.

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. During acute, lytic phase of infection, viral DNA can also be detected in serum. In the present study, the diagnostic utility of EBV DNA detection and quantitation in serum in primary EBV infection was investigated. The level of EBV DNA in the serum of 98 immunocompetent patients aged 1-47 years with symptomatic, antibody-confirmed EBV primary infection was assessed using a quantitative real-time PCR assay. The association between viral load and time after onset of disease, age and clinical and laboratory data was investigated. Quantitative PCR detected EBV DNA in 93 of 98 samples (94.9%), and the measured viral loads ranged from 3.8 x 10(1) to 6.6 x 10(4) copies/ml. EBV DNA detection exhibited a sensitivity of 94.9% and a specificity of 97.4% for primary EBV infection. EBV DNA was always detectable until day 12 after onset of symptoms, whereas no further positive PCR results were found after a period of 22 days after onset of disease. Detection of EBV DNA also showed a clearer association with the clinical manifestation of disease than the presence of EBV specific VCA IgG antibodies of low avidity. EBV DNA load was found to correlate inversely with the time after onset of disease (P < 0.001), and higher viral load levels were detected in younger (P = 0.009) and in hospitalized patients (P = 0.038). The results indicate that real-time PCR is a reliable tool for diagnosis of primary EBV infection early in the course of disease. In addition, EBV DNA detection may serve as a useful diagnostic supplement in serologically indeterminate EBV infections.