The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.     

On the putative role of transient receptor potential cation channels in asthma

The mammalian transient receptor potential (TRP) superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC (' C anonical'), TRPV (' V anilloid'), TRPM (' M elastatin'), TRPP (' P olycystin'), TRPML (' M uco l ipin') and the TRPA (' A nkyrin') groups. Increasing evidence has accumulated during the previous few years that links TRP channels to the cause of several diseases or to critically influence and/or determine their progress. This review focuses on the possible role of TRP channels in the aetiology of asthmatic lung disease.     

Inhibition of tamoxifen's therapeutic benefit by tangeretin in mammary cancer

Tangeretin, a molecule present in citrus fruits and in certain 'natural' menopausal medications, is an effective tumour growth and invasion inhibitor in vitro of human MCF 7/6 breast cancer cells. However, when added to the drinking water of MCF 7/6 tumour-bearing mice it neutralises the beneficial tumour-suppressing effect of tamoxifen. Tangeretin reduces the number of natural killer cells. This may explain why the beneficial suppressive effect of tangeretin on MCF 7/6 cell proliferation in vitro is completely counteracted in vivo.     

Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis

Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4(-/-) mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4(-/-) mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction.     

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4

Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcepsilonRI in mast cells triggers the influx of calcium (Ca(2+)) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca(2+) influx in mast cells. Trpm4(-/-) bone marrow-derived mast cells had more Ca(2+) entry than did TRPM4(+/+) cells after FcepsilonRI stimulation. Consequently, Trpm4(-/-) bone marrow-derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4(-/-) mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca(2+) entry in mast cells.     

Intraneuronal amyloid beta and reduced brain volume in a novel APP T714I mouse model for Alzheimer's disease

Transgenic mouse models of Alzheimer's disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.     

SUR1‐TRPM4 and AQP4 form a heteromultimeric complex that amplifies ion/water osmotic coupling and drives astrocyte swelling

Astrocyte swelling occurs after central nervous system injury and contributes to brain swelling, which can increase mortality. Mechanisms proffered to explain astrocyte swelling emphasize the importance of either aquaporin‐4 (AQP4), an astrocyte water channel, or of Na⁺‐permeable channels, which mediate cellular osmolyte influx. However, the spatio‐temporal functional interactions between AQP4 and Na⁺‐permeable channels that drive swelling are poorly understood. We hypothesized that astrocyte swelling after injury is linked to an interaction between AQP4 and Na⁺‐permeable channels that are newly upregulated. Here, using co‐immunoprecipitation and Förster resonance energy transfer, we report that AQP4 physically co‐assembles with the sulfonylurea receptor 1—transient receptor potential melastatin 4 (SUR1‐TRPM4) monovalent cation channel to form a novel heteromultimeric water/ion channel complex. In vitro cell‐swelling studies using calcein fluorescence imaging of COS‐7 cells expressing various combinations of AQP4, SUR1, and TRPM4 showed that the full tripartite complex, comprised of SUR1‐TRPM4‐AQP4, was required for fast, high‐capacity transmembrane water transport that drives cell swelling, with these findings corroborated in cultured primary astrocytes. In a murine model of brain edema involving cold‐injury to the cerebellum, we found that astrocytes newly upregulate SUR1‐TRPM4, that AQP4 co‐associates with SUR1‐TRPM4, and that genetic inactivation of the solute pore of the SUR1‐TRPM4‐AQP4 complex blocked in vivo astrocyte swelling measured by diolistic labeling, thereby corroborating our in vitro functional studies. Together, these findings demonstrate a novel molecular mechanism involving the SUR1‐TRPM4‐AQP4 complex to account for bulk water influx during astrocyte swelling. These findings have broad implications for the understanding and treatment of AQP4‐mediated pathological conditions.