Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA099

Poster Communications

Novel roles for the TRPM5 ion channel in regulating airway mucociliary clearance

M. I. Hollenhorst1, M. Empting2, H. Kanj1, P. Kumar1, V. Chubanov3, S. Appenzeller4, E. Saliba5, A. Hirsch2, G. Krasteva-Christ1

1. Institute of Anatomy and Cell Biology, Saarland University Faculty of Medicine, Homburg, Germany. 2. Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany. 3. Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilians-University, Munich, Germany. 4. Comprehensive Cancer Center, Biocenter, Julius-Maximilians-University, Wuerzburg, Germany. 5. Helmholtz Institute for RNA-based Infection Research, Wuerzburg, Germany.

Mucociliary clearance (MC) is a major innate defense mechanism that removes pathogens from the airways. It is dependent on concerted ciliary beating and transepithelial ion transport. Cholinergic chemosensory brush cells are airway epithelial cells that express components of the canonical bitter taste signaling cascade such as bitter taste receptors, Phospholipase b2 (PLCb2) and the TRPM5 (transient receptor potential subfamily M channel 5) ion channel. We showed previously that these cells sense "bitter tasting" bacterial products and bitter substances, and evoke protective respiratory reflexes utilizing the canonical taste transduction cascade. We here investigated the transcriptome of single tracheal ciliated and brush cells by single cell deep sequencing. The impact of various products from Pseudomonas aeruginosa (PA) on MC was estimated by the cilia-driven particle transport speed (PTS) on explanted mouse tracheas. PTS was visualized by tracking the transport of dynabeads on the surface of the mucosa before and after application of six PA products: PQS (3,4-dihydroxy-2-heptylquinoline, pseudomonas quinolone signal), 2-AA (2-aminoacetophenone), DHQ (2,4-dihydroxyquinoline), HHQ (4-hydroxy-2-heptylquinoline), HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide), and PyOC (pyocyanine). In addition, the influence of PQS and the bitter cascade agonist denatonium on transepithelial ion transport was examined with Ussing chamber experiments in murine isolated trachea. The effect on MC differed between the investigated PA products. PQS, 2-AA and DHQ (all 100 µM) significantly increased the PTS (p<0.0001, p=0.0004, p=0.0217, respectively). The most prominent increase was observed with PQS. NGS of single tracheal epithelial cells revealed TRPM5 and PLCβ2 as hallmark genes for brush cells. The PQS-induced increase was abolished with the TRPM5 antagonist TPPO (triphenylphosphine oxide, 100 µM) and significantly reduced in TRPM5-deficient mice (p=0.05). HHQ and HQNO (both 100 µM) decreased PTS (p=0.0159, p=0.0005, respectively), while PyOC (100 µM) had no effect. Furthermore, PQS (100 µM, apical) transiently increased net transepithelial ion current (Isc) (p=0.03), whereas denatonium (1 mM) decreased Isc (p<0.001). This effect was dose-dependent and repeatable without affecting the amplitude of the response. TPPO (basolateral, 100 µM) significantly reduced the effect of denatonium (1 mM, p=0.011), indicating an activation of the bitter taste signaling cascade involving TRPM5 activation by denatonium. Here, we show that bitter "tasting" PA products alter both major components of MC, the transepithelial ion transport and the cilia-mediated PTS. Both are dependent on activation of the TRPM5 channel present only in chemosensory brush cells. Thus, the TRPM5 ion channel is a novel key player in regulation of MC and might play an important role in protection against bacterial colonization.

Where applicable, experiments conform with Society ethical requirements