Mg2+-dependent Gating and Strong Inward Rectification of the Cation Channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca2+-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg2+. Mg2+ blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg2+ is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg2+, outward conductance is still ∼10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg2+-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg2+ sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg2+. The effects of intracellular Mg2+ on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K+ channels and may represent a novel regulatory mechanism for TRPV6 function in vivo

Parallel Selection on TRPV6 in Human Populations

We identified and examined a candidate gene for local directional selection in Europeans, TRPV6, and conclude that selection has acted on standing genetic variation at this locus, creating parallel soft sweep events in humans. A novel modification of the extended haplotype homozygosity (EHH) test was utilized, which compares EHH for a single allele across populations, to investigate the signature of selection at TRPV6 and neighboring linked loci in published data sets for Europeans, Asians and African-Americans, as well as in newly-obtained sequence data for additional populations. We find that all non-African populations carry a signature of selection on the same haplotype at the TRPV6 locus. The selective footprints, however, are significantly differentiated between non-African populations and estimated to be younger than an ancestral population of non-Africans. The possibility of a single selection event occurring in an ancestral population of non-Africans was tested by simulations and rejected. The putatively-selected TRPV6 haplotype contains three candidate sites for functional differences, namely derived non-synonymous substitutions C157R, M378V and M681T. Potential functional differences between the ancestral and derived TRPV6 proteins were investigated by cloning the ancestral and derived forms, transfecting cell lines, and carrying out electrophysiology experiments via patch clamp analysis. No statistically-significant differences in biophysical channel function were found, although one property of the protein, namely Ca2+ dependent inactivation, may show functionally relevant differences between the ancestral and derived forms. Although the reason for selection on this locus remains elusive, this is the first demonstration of a widespread parallel selection event acting on standing genetic variation in humans, and highlights the utility of between population EHH statistics

Cancer risk in immune-mediated inflammatory diseases (IMID).

Inflammation and cancer have a profound yet ambiguous relationship. Inflammation - especially chronic inflammation - has protumorigenic effects, but inflammatory cells also mediate an immune response against the tumor and immunosuppression is known to increase the risk for certain tumors.This article reviews current literature on the role of inflammation in cancer and the cancer risk in immune-mediated inflammatory diseases (IMIDs). We discuss the effect on cancer risk of different drug classes used in the treatment of IMIDs treatment, including biologicals such as tumor necrosis factor (TNF) inhibitors.Overall cancer incidence and mortality risk are similar to the general population in inflammatory bowel disease (IBD), and slightly increased for rheumatoid arthritis and psoriasis, with risk profiles differing for different tumor types. Increased risk for non-melanoma skin cancer is associated with thiopurine treatment in IBD, with the combination of anti-TNF and methotrexate in rheumatoid arthritis and with PUVA, cyclosporine and anti-TNF treatment in psoriasis. Data on the safety of using biologic or immunosuppressant therapy in IMID patients with a history of cancer are scarce.This review provides clinicians with a solid background to help them in making decisions about treatment of immune-mediated diseases in patients with a tumor history.This article is related to another review article in Molecular Cancer: http://www.molecular-cancer.com/content/12/1/86.Peer reviewe

RNA based approaches to profile oncogenic pathways from low quantity samples to drive precision oncology strategies

Precision treatment of cancer requires knowledge on active tumor driving signal transduction pathways to select the optimal effective targeted treatment. Currently only a subset of patients derive clinical benefit from mutation based targeted treatment, due to intrinsic and acquired drug resistance mechanisms. Phenotypic assays to identify the tumor driving pathway based on protein analysis are difficult to multiplex on routine pathology samples. In contrast, the transcriptome contains information on signaling pathway activity and can complement genomic analyses. Here we present the validation and clinical application of a new knowledge-based mRNA-based diagnostic assay platform (OncoSignal) for measuring activity of relevant signaling pathways simultaneously and quantitatively with high resolution in tissue samples and circulating tumor cells, specifically with very small specimen quantities. The approach uses mRNA levels of a pathway\u27s direct target genes, selected based on literature for multiple proof points, and used as evidence that a pathway is functionally activated. Using these validated target genes, a Bayesian network model has been built and calibrated on mRNA measurements of samples with known pathway status, which is used next to calculate a pathway activity score on individual test samples. Translation to RT-qPCR assays enables broad clinical diagnostic applications, including small analytes. A large number of cancer samples have been analyzed across a variety of cancer histologies and benchmarked across normal controls. Assays have been used to characterize cell types in the cancer cell microenvironment, including immune cells in which activated and immunotolerant states can be distinguished. Results support the expectation that the assays provide information on cancer driving signaling pathways which is difficult to derive from next generation DNA sequencing analysis. Current clinical oncology applications have been complementary to genomic mutation analysis to improve precision medicine: (1) prediction of response and resistance to various therapies, especially targeted therapy and immunotherapy; (2) assessment and monitoring of therapy efficacy; (3) prediction of invasive cancer cell behavior and prognosis; (4) measurement of circulating tumor cells. Preclinical oncology applications lie in a better understanding of cancer behavior across cancer types, and in development of a pathophysiology-based cancer classification for development of novel therapies and precision medicine

TRPP2 and TRPV4 form a polymodal sensory channel complex

The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing constitutes the fundamental mechanism of cystogenesis

TRPV Channels and Modulation by Hepatocyte Growth Factor/Scatter Factor in Human Hepatoblastoma (HepG2) Cells

Using patch clamp and Ca2+ imaging techniques, we have studied Ca2+ entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 μM), 4α-phorbol-12,13-didecanoate (4αPDD, 1 μM), arachidonic acid (10 μM), hypotonic stress, and heat all stimulated increases in [Ca2+]i within minutes. The increase in [Ca2+]i depended on extracellular Ca2+ and on the transmembrane potential, which indicated that both driving forces affected Ca2+ entry. Capsaicin also stimulated an increase in [Ca2+]i in nominally Ca2+-free solutions, which was compatible with the receptor functioning as a Ca2+ release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca2+ entry. Ca2+ influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca2+]i was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca2+-free solutions. 4αPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca2+ entry pathways in HepG2 cells. HGF/SF increases Ca2+ entry via TRPV1, but not via TRPV4. This rise in [Ca2+]i may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype

Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators

The transient receptor potential channel of the ankyrin-binding repeat subfamily, TRPA1, is a Ca2+-permeable non-selective cation channel that depolarizes the plasma membrane and causes Ca2+ influx. A typical feature of TRPA1 is its rapid desensitization following activation by agonists such as mustard oil (MO), cinnamaldehyde, and a high intracellular Ca2+ concentration. In whole-cell recordings on Chinese hamster ovary (CHO) cells expressing TRPA1, desensitization was delayed when phosphatidylinositol 4,5-biphosphate (PIP2) was supplemented via the patch pipette, whereas the PIP2 scavenger neomycin accelerated desensitization. Preincubation with the PI-4 kinase inhibitor wortmannin reduced both constitutive TRPA1 channels activity and the response to MO. Run down was also accelerated by high intracellular Mg2+ concentrations, whereas chelating intracellular Mg2+ with 10 mM ethylenedinitrilotetraacetic acid (EDTA) increased the basal channel activity. In inside-out patches, we observed a rapid run down of TRPA1 activity, which could be prevented by application of diC8-PIP2 or 2 mM Mg-ATP but not Na2-ATP to the cytosolic side of the excised patches. In isolated trigeminal ganglion neurons, preincubation with wortmannin resulted in inhibition of endogenous TRPA1 activation by MO. Taken together, our data indicate that PIP2 modulates TRPA1, albeit to a lesser extent than other known PIP 2-dependent TRP channels, and that tools modifying the plasma membrane PIP2 content often have direct effects on this channel. © 2008 Springer-Verlag.This work was supported by the Human Frontiers Science Programme (HFSP Research Grant Ref. RGP 32/2004), the Belgian Federal Government, the Flemish Government, the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0214.99, F.W.O. G. 0136.00; F.W.O. G.0172.03, Interuniversity Poles of Attraction Program, Prime Ministers Office IUAP) and the Excellentiefinanciering (EF/95/010).Peer Reviewe

The selectivity filter of the cation channel TRPM4

Transient receptor potential channel melastatin subfamily (TRPM) 4 and its close homologue, TRPM5, are the only two members of the large transient receptor potential superfamily of cation channels that are impermeable to Ca(2+). In this study, we located the TRPM4 selectivity filter and investigated possible structural elements that render it Ca(2+)-impermeable. Based on homology with known cation channel pores, we identified an acidic stretch of six amino acids in the loop between transmembrane helices TM5 and TM6 ((981)EDMDVA(986)) as a potential selectivity filter. Substitution of this six-amino acid stretch with the selectivity filter of TRPV6 (TIIDGP) resulted in a functional channel that combined the gating hallmarks of TRPM4 (activation by Ca(2+), voltage dependence) with TRPV6-like sensitivity to block by extracellular Ca(2+) and Mg(2+) as well as Ca(2+) permeation. Neutralization of Glu(981) resulted in a channel with normal permeability properties but a strongly reduced sensitivity to block by intracellular spermine. Neutralization of Asp(982) yielded a functional channel that exhibited extremely fast desensitization (tau < 5 s), possibly indicating destabilization of the pore. Neutralization of Asp(984) resulted in a non-functional channel with a dominant negative phenotype when coexpressed with wild type TRPM4. Combined neutralization of all three acidic residues resulted in a functional channel whose voltage dependence was shifted toward very positive potentials. Substitution of Gln(977) by a glutamate, the corresponding residue in divalent cation-permeable TRPM channels, altered the monovalent cation permeability sequence and resulted in a pore with moderate Ca(2+) permeability. Our findings delineate the selectivity filter of TRPM channels and provide the first insight into the molecular basis of monovalent cation selectivity.status: publishe