TRPC6 channel translocation into phagosomal membrane augments phagosomal function.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

Cell-based potassium ion channel screening using the FluxOR assay.

FluxOR technology is a cell-based assay used for high-throughput screening measurements of potassium channel activity. Using thallium influx as a surrogate indicator of potassium ion channel activity, the FluxOR Potassium Ion Channel Assay is based on the activation of a novel fluorescent dye. This indicator reports channel activity with a large fluorogenic response and is proportional to the number of open potassium channels on the cell, making it extremely useful for studying K(+) channel targets. In contrast to BTC-AM ester, FluxOR dye is roughly 10-fold more thallium sensitive, requiring much lower thallium for a larger signal window. This also means that the assay is carried out in a physiological, normal-chloride saline. In this article, the authors describe how they used BacMam gene delivery to express Kv7.2 and 7.3 (KCNQ), Kir2.1, or Kv11.1 (hERG) potassium ion channels in U2-OS cells. Using these cells, they ran the FluxOR assay to identify and characterize channel-specific inhibitory compounds discovered within the library (Tocriscreen Mini 1200 and Sigma Sodium/Potassium Modulators Ligand set). The FluxOR assay was able to identify several known specific inhibitors of Kv7.2/7.3 or hERG, highlighting its potential to identify novel and more efficacious small-molecule modulators.

Disease-causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages.

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

Quantitative single-cell differences in mu-opioid receptor mRNA distinguish myelinated and unmyelinated nociceptors.

A remarkable feature of opioids is that they inhibit pain that persists from previous injuries without eliminating either the initial pain of a new injury or the protective reflexes triggered by it. Here we ask whether selective expression of the mu-opioid receptor (MOR) gene in primary nociceptors (pain-sensing neurons) might contribute to this aspect of opioid specificity. We quantified single-cell levels of MOR mRNA and measured opioid inhibition of Ca channels on identified nociceptors and low-threshold mechanosensors (non-nociceptors) isolated from rats. Negligibly few non-nociceptors express MOR mRNA, thereby rendering nonpain sensations insensitive to opioids. Nearly half of nociceptors of all size classes also fail to express MOR mRNA or to respond to opioids. Among the opioid-responsive nociceptors, a gene dose-response relationship exists such that maximal opioid inhibition occurs when the MOR mRNA concentration of a cell is >15 pm. Almost all large, myelinated nociceptors express MOR mRNA below this level, whereas small, unmyelinated nociceptors are likely to express above it. Because myelinated nociceptors mediate anti-nociceptive reflexes, the data suggest that fine control of the MOR mRNA level contributes to a complex neural trait: the ability of opioids to suppress persistent pain without preventing response to a new injury.