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Introduction Constant exposure of the airways to inhaled particles and pathogens requires efficient early immune responses protecting against infections. These processes are largely mediated by the airway epithelium that is [...], Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene TrpmS, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into firstline defense mechanisms against bacterial infections of the lung.

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER–PM junctions) as shown by colocalization with ER–PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER–PM junctional pool of PI(4,5)P2. Finally, we found that association with ER–PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs [ABSTRACT FROM AUTHOR]

Mucociliary clearance is a primary defence mechanism of the airways consisting of two components, ciliary beating and transepithelial ion transport (ISC). Specialised chemosensory cholinergic epithelial cells, named brush cells (BC), are involved in regulating various physiological and immunological processes. However, it remains unclear if BC influence ISC. In murine tracheae, denatonium, a taste receptor agonist, reduced basal ISC in a concentration-dependent manner (EC50 397 µM). The inhibition of bitter taste signalling components with gallein (Gβγ subunits), U73122 (phospholipase C), 2-APB (IP3-receptors) or with TPPO (Trpm5, transient receptor potential-melastatin 5 channel) reduced the denatonium effect. Supportively, the ISC was also diminished in Trpm5−/− mice. Mecamylamine (nicotinic acetylcholine receptor, nAChR, inhibitor) and amiloride (epithelial sodium channel, ENaC, antagonist) decreased the denatonium effect. Additionally, the inhibition of Gα subunits (pertussis toxin) reduced the denatonium effect, while an inhibition of phosphodiesterase (IBMX) increased and of adenylate cyclase (forskolin) reversed the denatonium effect. The cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh172 and the KCNQ1 potassium channel antagonist chromanol 293B both reduced the denatonium effect. Thus, denatonium reduces ISC via the canonical bitter taste signalling cascade leading to the Trpm5-dependent nAChR-mediated inhibition of ENaC as well as Gα signalling leading to a reduction in cAMP-dependent ISC. Therefore, BC activation contributes to the regulation of fluid homeostasis. [ABSTRACT FROM AUTHOR]

The three members of the ether-à-go-go-gene-like (Elk; Kv12.1-Kv12.3) family of voltage-gated K+ channels are predominantly expressed in neurons, but only little information is available on their physiological relevance. It was shown that Kv12.2 channels modulate excitability of hippocampal neurons, but no native current could be attributed to Kv12.1 and Kv12.3 subunits yet. This may appear somewhat surprising, given high expression of their mRNA transcripts in several brain areas. Native Kv12 currents may have been overlooked so far due to limited knowledge on their biophysical properties and lack of specific pharmacology. Except for Kv12.2, appropriate genetically modified mouse models have not been described; therefore, identification of Kv12-mediated currents in native cell types must rely on characterization of unique properties of the channels. We focused on recombinant human Kv12.1 to identify distinct properties of these channels. We found that Kv12.1 channels exhibited significant mode shift of activation, i.e., stabilization of the voltage sensor domain in a "relaxed" open state after prolonged channel activation. This mode shift manifested by a slowing of deactivation and, most prominently, a significant shift of voltage dependence to hyperpolarized potentials. In contrast to related Kv11.1, mode shift was not sensitive to extracellular Na+, which allowed for discrimination between these isoforms. Sensitivity of Kv12.1 and Kv11.1 to the broad-spectrum K+ antagonist 4-aminopyridine was similar. However, 4-AP strongly activated Kv12.1 channels, but it was an inhibitor of Kv11 channels. Interestingly, the agonist of Kv11 channels NS1643 also differentially modulated the activity of these channels, i.e., NS1643 activated Kv11.1, but strongly inhibited Kv12.1 channels. Thus, these closely related channels are distinguished by inverse pharmacological profiles. In summary, we identified unique biophysical and pharmacological properties of Kv12.1 channels and established straightforward experimental protocols to characterize Kv12.1-mediated currents. Identification of currents in native cell types with mode shift that are activated through 4-AP and inhibited by NS1643 can provide strong evidence for contribution of Kv12.1 to whole cell currents. [ABSTRACT FROM AUTHOR]

Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

The three members of the ether-à-go-go-gene -like (Elk; K v 12.1-K v 12.3) family of voltage-gated K + channels are predominantly expressed in neurons, but only little information is available on their physiological relevance. It was shown that K v 12.2 channels modulate excitability of hippocampal neurons, but no native current could be attributed to K v 12.1 and K v 12.3 subunits yet. This may appear somewhat surprising, given high expression of their mRNA transcripts in several brain areas. Native K v 12 currents may have been overlooked so far due to limited knowledge on their biophysical properties and lack of specific pharmacology. Except for K v 12.2, appropriate genetically modified mouse models have not been described; therefore, identification of K v 12-mediated currents in native cell types must rely on characterization of unique properties of the channels. We focused on recombinant human K v 12.1 to identify distinct properties of these channels. We found that K v 12.1 channels exhibited significant mode shift of activation, i.e., stabilization of the voltage sensor domain in a "relaxed" open state after prolonged channel activation. This mode shift manifested by a slowing of deactivation and, most prominently, a significant shift of voltage dependence to hyperpolarized potentials. In contrast to related K v 11.1, mode shift was not sensitive to extracellular Na + , which allowed for discrimination between these isoforms. Sensitivity of K v 12.1 and K v 11.1 to the broad-spectrum K + antagonist 4-aminopyridine was similar. However, 4-AP strongly activated K v 12.1 channels, but it was an inhibitor of K v 11 channels. Interestingly, the agonist of K v 11 channels NS1643 also differentially modulated the activity of these channels, i.e., NS1643 activated K v 11.1, but strongly inhibited K v 12.1 channels. Thus, these closely related channels are distinguished by inverse pharmacological profiles. In summary, we identified unique biophysical and pharmacological properties of K v 12.1 channels and established straightforward experimental protocols to characterize K v 12.1-mediated currents. Identification of currents in native cell types with mode shift that are activated through 4-AP and inhibited by NS1643 can provide strong evidence for contribution of K v 12.1 to whole cell currents.