The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

The epithelial Ca2+ channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca2+ reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845–2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca2+-concentrations

RyRCa2+ Leak Limits Cardiac Ca2+ Window Current Overcoming the Tonic Effect of Calmodulin in Mice

Ca2+ mediates the functional coupling between L-type Ca2+ channel (LTCC) and sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca2+-induced Ca2+-release (CICR) mechanism triggered by Ca2+ influx, but also as the retrograde Ca2+-dependent inactivation (CDI) of LTCC, which depends on both Ca2+ permeating through the LTCC itself and on SR Ca2+ release through the RyR. This latter effect has been suggested to rely on local rather than global Ca2+ signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca2+ leak, we evidence here that increased occurrence of the discrete local SR Ca2+ releases through the RyRs (Ca2+ sparks) causea depolarizing shift in activation and a hyperpolarizing shift inisochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca2+]i buffer capacity or depleting SR Ca2+ store blunted these changes, which could be reproduced in WT cells by RyRCa2+ leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca2+ control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca2+ signals and CaM function

Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca2+ in Fission Yeast

The regulation of cytoplasmic Ca2+ is crucial for various cellular processes. Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state

Tumour gene expression signature in primary melanoma predicts long-term outcomes

Adjuvant systemic therapies are now routinely used following resection of stage III melanoma, however accurate prognostic information is needed to better stratify patients. We use differential expression analyses of primary tumours from 204 RNA-sequenced melanomas within a large adjuvant trial, identifying a 121 metastasis-associated gene signature. This signature strongly associated with progression-free (HR = 1.63, p = 5.24 × 10−5) and overall survival (HR = 1.61, p = 1.67 × 10−4), was validated in 175 regional lymph nodes metastasis as well as two externally ascertained datasets. The machine learning classification models trained using the signature genes performed significantly better in predicting metastases than models trained with clinical covariates (pAUROC = 7.03 × 10−4), or published prognostic signatures (pAUROC < 0.05). The signature score negatively correlated with measures of immune cell infiltration (ρ = −0.75, p < 2.2 × 10−16), with a higher score representing reduced lymphocyte infiltration and a higher 5-year risk of death in stage II melanoma. Our expression signature identifies melanoma patients at higher risk of metastases and warrants further evaluation in adjuvant clinical trials

Activation of the hypothalamic-pituitary-adrenal axis by exogenous and endogenous GDF15

\ua9 2021 National Academy of Sciences. All rights reserved.An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15−/− mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies