TRPC6 channel translocation into phagosomal membrane augments phagosomal function

Abstract

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.

Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis

“…Adapted from ref. [79]. Bacterial growth is prevented by cftr +/+ AM (data not shown), but not by F508del-CFTR or cftr−/− AM.…”

“…Once in the organism roscovitine is rapidly metabolized by the liver, essentially by oxidation [27]. The main metabolite is the carboxylate product (M3) (Figure 1) [66, 67, 73, 77], which does not inhibit the kinases targeted by roscovitine but may account for other effects of the drug [77–79]. Although the half-life of M3 in humans is similar to that of roscovitine [66, 67], its CF favorable biological activity (see below) could extend that of roscovitine in CF treatment.…”

“…Roscovitine could therefore have a positive bactericidal effect on most CF forms. Improvement of the bactericidal properties is observed with the M3 metabolite, S-CR8, N6-methyl-roscovitine and O6-benzyl-roscovitine, but is not observed with S-roscovitine, miglustat, olomoucine, finisterine, perharidine or purvalanol A [79]. …”

“…Recent results suggest that the effects of roscovitine on the intra-phagolysosomal pH of macrophages could be explained by an action mediated by the Ca 2+ permeable channel TRPC6 [79]. TRPC6 belongs to the ‘Transient Receptor Potential’ (TRP) family of ion channels, particularly important in respiratory system diseases.…”

From Starfish Oocytes to Inflammation: The Unforeseeable Destiny of Roscovitine in Cystic Fibrosis

“…This high intraphagolysosomal pH prevents lysosomal proteases and lipases from digesting phagocytosed bacteria, and this may explain the high rate of bacterial infections in the lungs of CF patients. Roscovitine was tested for its potential effect on intraphagolysosomal pH and surprisingly restored the acidic ability of phagolysosomes to destroy bacteria, ultimately improving the bactericidal activity of CFTR-deficient alveolar macrophages [17]. However, this effect was not due to any correcting effect of roscovitine on F508del-CFTR since identical results were obtained in the complete absence of CFTR (cftr-/- macrophages).…”