Activation of TRPM7 channels by small molecules under physiological conditions.

Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a cation channel covalently linked to a protein kinase domain. TRPM7 is ubiquitously expressed and regulates key cellular processes such as Mg(2+) homeostasis, motility, and proliferation. TRPM7 is involved in anoxic neuronal death, cardiac fibrosis, and tumor growth. The goal of this work was to identify small molecule activators of the TRPM7 channel and investigate their mechanism of action. We used an aequorin bioluminescence-based assay to screen for activators of the TRPM7 channel. Valid candidates were further characterized using patch clamp electrophysiology. We identified 20 drug-like compounds with various structural backbones that can activate the TRPM7 channel. Among them, the δ opioid antagonist naltriben was studied in greater detail. Naltriben's action was selective among the TRP channels tested. Naltriben activates TRPM7 currents without prior depletion of intracellular Mg(2+) even under conditions of low PIP2. Moreover, naltriben interfered with the effect of the TRPM7 inhibitor NS8593. Finally, our experiments with TRPM7 variants carrying mutations in the pore, TRP, and kinase domains indicate that the site of TRPM7 activation by this small-molecule ligand is most likely located in or near the TRP domain. In conclusion, we identified the first organic small-molecule activators of TRPM7 channels, thus providing new experimental tools to study TRPM7 function in native cellular environments.

Cholinergic chemosensory cells in the auditory tube.

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(ß2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCß2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

The role of classical transient receptor potential channels in the regulation of hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung matching blood perfusion to ventilation during local alveolar hypoxia. HPV thus optimizes pulmonary gas exchange. In contrast chronic and generalized hypoxia leads to pulmonary vascular remodeling with subsequent pulmonary hypertension and right heart hypertrophy. Among other non-selective cation channels, the family of classical transient receptor potential channels (TRPC) has been shown to be expressed in pulmonary arterial smooth muscle cells. Among this family, TRPC6 is essential for the regulation of acute HPV in mice. Against this background, in this chapter we give an overview about the TRPC family and their role in HPV.

Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms.

Hypoxic pulmonary vasoconstriction (HPV), also known as the von Euler-Liljestrand mechanism, is a physiological response to alveolar hypoxia which distributes pulmonary capillary blood flow to alveolar areas of high oxygen partial pressure. Impairment of this mechanism may result in hypoxaemia. Under conditions of chronic hypoxia generalised vasoconstriction of the pulmonary vasculature in concert with hypoxia-induced vascular remodelling leads to pulmonary hypertension. Although the principle of HPV was recognised decades ago, its exact pathway still remains elusive. Neither the oxygen sensing process nor the exact pathway underlying HPV is fully deciphered yet. The effector pathway is suggested to include L-type calcium channels, nonspecific cation channels and voltage-dependent potassium channels, whereas mitochondria and nicotinamide adenine dinucleotide phosphate oxidases are discussed as oxygen sensors. Reactive oxygen species, redox couples and adenosine monophosphate-activated kinases are under investigation as mediators of hypoxic pulmonary vasoconstriction. Moreover, the role of calcium sensitisation, intracellular calcium stores and direction of change of reactive oxygen species is still under debate. In this context the present article focuses on the basic mechanisms of hypoxic pulmonary vasoconstriction and also outlines differences in current concepts that have been suggested for the regulation of hypoxic pulmonary vasoconstriction.

Loss of classical transient receptor potential 6 channel reduces allergic airway response.

BACKGROUND: Non-selective cation influx through canonical transient receptor potential channels (TRPCs) is thought to be an important event leading to airway inflammation. TRPC6 is highly expressed in the lung, but its role in allergic processes is still poorly understood. OBJECTIVE: The purpose of this study was to evaluate the role of TRPC6 in airway hyperresponsiveness (AHR) and allergic inflammation of the lung. METHODS: Methacholine-induced AHR was assessed by head-out body plethysmography of wild type (WT) and TRPC6(-/-) mice. Experimental airway inflammation was induced by intraperitoneal ovalbumin (OVA) sensitization, followed by OVA aerosol challenges. Allergic inflammation and mucus production were analysed 24 h after the last allergen challenge. RESULTS: Methacholine-induced AHR and agonist-induced contractility of tracheal rings were increased in TRPC6(-/-) mice compared with WT mice, most probably due to compensatory up-regulation of TRPC3 in airway smooth muscle cells. Most interestingly, when compared with WT mice, TRPC6(-/-) mice exhibited reduced allergic responses after allergen challenge as evidenced by a decrease in airway eosinophilia and blood IgE levels, as well as decreased levels of T-helper type 2 (Th2) cytokines (IL-5, IL-13) in the bronchoalveolar lavage. However, lung mucus production after allergen challenge was not altered by TRPC6 deficiency. CONCLUSIONS: TRPC6 deficiency inhibits specific allergic immune responses, pointing to an important immunological function of this cation channel in Th2 cells, eosinophils, mast cells and B cells.

Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer. Molecular basis and clinical implication.

Recent studies with transfected COS-7 cells have shown that functionally inactive mutant V2 vasopressin receptors (occurring in patients with nephrogenic diabetes insipidus) can be functionally rescued by coexpression of a carboxy-terminal V2 receptor fragment (V2-tail) spanning the region where various mutations occur [Schöneberg, T., J. Yun, D. Wenkert, and J. Wess. 1996. EMBO (Eur. Mol. Biol. Organ.) J. 15:1283-1291]. In this study, we set out to characterize the underlying molecular mechanism. Using a coimmunoprecipitation strategy and a newly developed sandwich ELISA system, a direct and highly specific interaction between the mutant V2 vasopressin receptor proteins and the V2-tail polypeptide was demonstrated. To study the potential therapeutic usefulness of these findings, Chinese hamster ovary (CHO) cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors were created and infected with a recombinant adenovirus carrying the V2-tail gene fragment. After adenovirus infection, vasopressin gained the ability to stimulate cAMP formation with high potency and efficacy in all CHO cell clones studied. Moreover, adenovirus-mediated gene transfer also proved to be a highly efficient method for achieving expression of the V2-tail fragment (as well as the wild-type V2 receptor) in Madin-Darby canine kidney tubular cells. Taken together, these studies clarify the molecular mechanisms by which receptor fragments can restore function of mutationally inactivated G protein-coupled receptors and suggest that adenovirus-mediated expression of receptor fragments may lead to novel strategies for the treatment of a variety of human diseases.

TRPC6.

TRPC6 is a Ca(2+)-permeable non-selective cation channel expressed in brain, smooth muscle containing tissues and kidney, as well as in immune and blood cells. Channel homomers heterologously expressed have a characteristic doubly rectifying current-voltage relationship and are six times more permeable for Ca2+ than for Na+. In smooth muscle tissues, however, Na+ influx and activation of voltage-gated calcium channels by membrane depolarization rather than Ca2+ elevation by TRPC6 channels is the driving force for contraction. TRPC6 channels are directly activated by the second messenger diacylglycerol (DAG) and regulated by specific tyrosine or serine phosphorylation. Extracellular Ca2+ has inhibitory effects, while Ca2+/calmodulin acting from the intracellular side has potentiator effects on channel activity. Given its specific expression, TRPC6 is likely to play a number of physiological roles. Studies with TRPC6(-/-) mice suggest a role for the channel in the regulation of vascular and pulmonary smooth muscle contraction. TRPC6 was identified as an essential component of the slit diaphragm architecture of kidney podocytes. Other functions in immune and blood cells, as well as in brain and in smooth muscle-containing tissues such as stomach, colon and myometrium, remain elusive.

The novel MKL target gene myoferlin modulates expansion and senescence of hepatocellular carcinoma.

Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are transcriptional coactivators of Serum Response Factor (SRF) with an essential role for hepatocellular carcinoma (HCC) growth and oncogene-induced senescence. In this report, we identified myoferlin as a novel MKL/SRF target gene by gene expression profiling and verification in vivo in HCC xenografts. Myoferlin was overexpressed in human and murine HCCs triggered by conditional expression of constitutively active SRF-VP16 protein in hepatocytes. Furthermore, myoferlin was required for HCC cell invasion, proliferation and anchorage-independent cell growth. We provide evidence that myoferlin is a crucial gene target of MKL1/2 mediating its effect on oncogene-induced senescence by modulating the activation state of the EGFR and downstream MAPK and p16-/Rb pathways. Depletion of myoferlin in tumour cells from SRF-VP16-derived murine HCCs induced a senescence phenotype. These findings identify MKL1/2 and myoferlin as novel therapeutic targets to treat human HCC by a senescence-inducing strategy.

The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to G(q), G(i) and G(12) proteins.

Neuropeptides like galanin produced and released by small cell lung cancer (SCLC) cells are considered principal mitogens in these tumors. We identified the galanin receptor type 2 (GALR2) as the only galanin receptor expressed in H69 and H510 cells. Photoaffinity labeling of G proteins in H69 cell membranes revealed that GALR2 activates G proteins of three subfamilies: G(q), G(i), and G(12). In H69 cells, galanin-induced Ca2+ mobilization was pertussis toxin-insensitive. While phorbol ester-induced extracellular signal-regulated kinase (ERK) activation required protein kinase C (PKC) activity, preincubation of H69 cells with the PKC-inhibitor GF109203X had no effect on galanin-dependent ERK activity. A rise of the intracellular calcium concentration was necessary and sufficient to mediate galanin-induced ERK activation. In support of G(i) coupling, stimulation of GALR2 expressed in HEK293 cells inhibited isoproterenol-induced cAMP accumulation and raised cAMP levels in COS-7 cells when coexpressed with a chimeric G alpha(S)-G alpha(i) protein In H69 cells, galanin activated the monomeric GTPase RhoA and induced stress fiber formation in Swiss 3T3 cells expressing GALR2. Thus, we provide the first direct evidence that in SCLC the mitogenic neuropeptide galanin, interacting with GALR2, simultaneously activates multiple classes of G proteins and signals through the G(q) phospholipase C/calcium sequence and a G(12)/Rho pathway. Oncogene (2000) 19, 4199 - 4209

A novel subgroup of class I G-protein-coupled receptors.

Based on structural similarities of an expressed sequence tag with the platelet-activating factor (PAF) receptor a cDNA clone encoding a novel G-protein-coupled receptor (GPCR), named GPR34, was isolated from a human fetal brain cDNA library. Genomic DNA analyses revealed the receptor to be encoded by an intronless single-copy gene at Xp11. 3-11.4. The predicted 381-amino-acid protein disclosed all structural features characteristic of a member of the class I GPCR family. Except an obvious sequence homology in transmembrane domain 6, no further similarities to the PAF receptor or any other known GPCR were found. The corresponding mouse receptor DNA was isolated from a genomic P1 library displaying a 90% amino acid identity compared to the human receptor. Phylogenetic studies showed that GPR34 is preserved among vertebrates, and the existence of GPR34 subtypes was demonstrated. The receptor mRNA is abundantly expressed in human and mouse tissues. In addition to the major 2-kb transcript, a 4-kb transcript was found only in mouse liver and testis. Expression of the human GPR34 in COS-7 cells followed by Western blot studies revealed specific bands of a highly glycosylated protein between 75 and 90 kDa. A number of potential ligands including phospholipids, leukotrienes, hydroxy-eicosatetraenoic acids, nucleotides and peptides were tested in functional assays. Since none of the applied substances led to significant changes in second messenger levels (cAMP and inositol phosphates), the natural ligand and coupling profile of this novel GPCR subgroup remains unknown.

Emerging roles of TRPM6/TRPM7 channel kinase signal transduction complexes.

Investigations into Drosophila mutants with impaired vision due to mutations in the transient receptor potential gene (trp) initiated a systematic search for TRP homologs in other species, finally leading to the discovery of a whole new family of plasma membrane cation channels involved in multiple physiological processes. Among the recently discovered TRP cation channels two homologous proteins, TRPM6 and TRPM7, display unique domain compositions and biophysical properties. These remarkable genes are vital for Mg(2+) homeostasis in vertebrates and, if disrupted, lead to cell death or human disease.

Hormone binding to the follicle-stimulating hormone receptor--crystal clear!

The receptors for the trophic hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) play a central role in endocrinology. These receptors face the challenge to accommodate large heterodimeric glycoprotein ligands within their extracellular hormone-binding domain. Until recently, the mechanism of hormone binding and consequently the mode of receptor activation remained enigmatic. By solving the crystal structure of human follicle-stimulating hormone bound to the receptor's hormone binding domain, it has become clear that the follicle-stimulating hormone receptor grabs the glycoprotein hormone in a hand-clasp mode resulting in a hormone orientation perpendicular to the long axis of the ligand-binding domain. These findings have important ramifications for our understanding of the molecular mechanism of receptor activation and may provide a rational basis for the development of small, non-peptidic FSH receptor ligands.

Receptors and G proteins as primary components of transmembrane signal transduction. Part 2. G proteins: structure and function.

Seven-transmembrane receptors signal through nucleotide-binding proteins (G proteins) into the cell. G proteins are membrane-associated proteins composed of three subunits termed alpha, beta and gamma, of which the G alpha subunit classifies the heterotrimer. So far, 23 different mammalian G alpha subunits are known, which are grouped in four subfamilies (Gs, Gi, Gq, G12) on the basis of their amino acid similarity. They carry an endogenous GTPase activity allowing reversible functional coupling between ligand-bound receptors and effectors such as enzymes and ion channels. In addition, five G beta and seven G gamma subunits have been identified which form tightly associated beta gamma heterodimers. Upon activation by a ligand-bound receptor the G protein dissociates into G alpha and G beta gamma, which both transmit signal by interacting with effectors. On the G protein level, specificity and selectivity of the incoming signal is accomplished by G protein trimers composed of distinct subunits. On the other hand, many receptors have been shown to activate different G proteins, thereby regulating diverse signal transduction pathways.

Transient receptor potential channels as molecular substrates of receptor-mediated cation entry.

Calcium is a versatile multitarget intracellular second messenger in eukaryotic cells. In addition to calcium release from intracellular stores and influx via voltage- or ligand-operated channels, agonist-induced calcium entry constitutes one of the main pathways by which cytosolic calcium is elevated. Receptor-stimulated currents are initiated in response to agonist binding to G-protein-coupled receptors and to receptor tyrosine kinases. Within the past few years our knowledge about the molecular identity of receptor-stimulated channels has expanded substantially. Drosophila melanogaster visual transduction channels associated with the transient receptor potential (trp) and the trp-like (trpl) mutant visual phenotypes were the first members of this category of channels to be identified at the molecular level. Since then an entire mammalian gene family of TRP homologues has been discovered by homology cloning. Only now are we beginning to fully understand the functional roles of TRP channels in mammalian cells. We review recent findings in TRP channel research and discuss the role of these proteins for receptor-activated cation entry.

Thyrotropin receptor mutations as a tool to understand thyrotropin receptor action.

A large number of mutations have been identified in the thyrotropin (TSH) receptor (TSHR) gene causing human diseases. Toxic thyroid nodules are frequently associated with somatic constitutively activating TSHR mutations. Autosomal dominant non-autoimmune hyperthyroidism is caused by activating TSHR germline mutations. Inactivating germline mutations cause TSH unresponsiveness. Discovery of the different TSHR mutations in various regions of the receptor molecule has led to the identification of important domains for intramolecular TSHR signal transduction. However, despite the functional characterization of the naturally occurring mutations the precise molecular mechanisms of receptor activation including the processes of hormone binding, intramolecular signaling between the different TSHR domains and of G protein coupling are not completely understood. This review discusses the importance of the various receptor domains for TSHR activation identified on the basis of the naturally occurring gain or loss of function mutations and in vitro investigations performed with site-directed mutagenesis, synthetic peptides, or antibodies. Several in vitro studies have provided new insights into structure-function relationships by site-directed mutagenesis in combination with molecular modeling. These in vitro investigations have often been guided by naturally occurring mutations and have provided new insights into intramolecular changes during receptor activation. This has led to progress in understanding the mechanism of TSHR activation.

Inhibition of gonadotropin-releasing hormone receptor signaling by expression of a splice variant of the human receptor.

GnRH binds to a specific G protein-coupled receptor in the pituitary to regulate synthesis and secretion of gonadotropins. Using RT-PCR and human pituitary poly(A)+ RNA as a template, the full-length GnRH receptor (wild type) and a second truncated cDNA characterized by a 128-bp deletion between nucleotide positions 522 and 651 were cloned. The deletion causes a frame shift in the open reading frame, thus generating new coding sequence for further 75 amino acids. The truncated cDNA arises from alternative splicing by accepting a cryptic splicing acceptor site in exon 2. Distinct translation products of approximately 45-50 and 42 kDa were immunoprecipitated from COS-7 cells transfected with cDNA coding for wild type GnRH receptor and the truncated splice variant, respectively. Immunocytochemical and enzyme-linked immunosorbent assay studies revealed a membranous expression pattern for both receptor isoforms. Expression of the splice variant, however, occurred at a significantly lower cell surface receptor density. In terms of ligand binding and phospholipase C activation, the wild type receptor showed characteristics of a typical GnRH receptor, whereas the splice variant was incapable of ligand binding and signal transduction. Coexpression of wild type and truncated proteins in transiently or stably transfected cells, however, resulted in impaired signaling via the wild type receptor by reducing maximal agonist-induced inositol phosphate accumulation. The inhibitory effect depended on the amount of splice variant cDNA cotransfected and was specific for the GnRH receptor because signaling via other G(q/11)-coupled receptors, such as the thromboxane A2, M5 muscarinic, and V1 vasopressin receptors, was not affected. Immunological studies revealed that coexpression of the wild type receptor and the truncated splice variant resulted in impaired insertion of the wild type receptor into the plasma membrane. Thus, expression of truncated receptor proteins may highlight a novel principle of specific functional inhibition of G protein-coupled receptors.

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C.

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.

Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3',5'-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways.

The cDNAs encoding the murine LH receptor (LHR) and the human beta 2-adrenoceptor (h beta 2AR) were cloned and RNAs complementary to their sense strands (cRNAs) were injected into defolliculated Xenopus oocytes. This led to expression, respectively, of LH- and isoproterenol-stimulable adenylyl cyclase activities, indicating that functionally active receptor cDNAs had been cloned. In oocytes injected with LHR cRNA, but not in control or h beta 2AR cRNA-injected oocytes, human CG and LH increased a Ca(2+)-activated Cl- current, as measured by the two-microelectrode voltage-clamp method. This effect was not seen with isoproterenol in control or h beta 2AR cRNA-injected oocytes, it was also not observed in response to forskolin or (Bu)2cAMP. The response to human CG could be obtained in the absence of extracellular Ca2+ but was abolished by injection of EGTA, indicating that it was caused by mobilization of Ca2+ from intracellular stores. The response was unaffected by overnight treatment with 1 microgram/ml pertussis toxin. The experiments show that a glycoprotein hormone receptor can be expressed as a functionally active molecule in Xenopus oocytes, and that the LHR has the ability of activating two separate intracellular signaling pathways: one forming the second messenger cAMP, and the other mobilizing Ca2+ from intracellular stores. It is proposed that the latter is secondary to a primary activation of phospholipase C by the LHR, which elevates intracellular Ca2+ via intermediary elevation of inositol phosphates, presumably (1,4,5)inositol trisphosphate.

Role of the third intracellular loop for the activation of gonadotropin receptors.

Hyperfunctional endocrine thyroid and testicular disorders can frequently be traced back to gainof-function mutations in glycoprotein hormone receptor genes. Deletion mutations in the third intracellular (i3) loop of the TSH receptor have recently been identified as a cause of constitutive receptor activity. To examine whether the underlying mechanism of receptor activation applies to all glycoprotein hormone receptors, we created deletion mutations in the LH and FSH receptors. In analogy to the situation with the TSH receptor, a deletion of nine amino acids resulted in constitutive activity irrespective of the location of deletions within the i3 loop of the LH receptor. In contrast, only one (delta563-566) of four different 4-amino acid deletion mutants displayed agonist-independent activity. Systematic examination of the structural requirements for this effect in the delta563-566 mutant revealed that only deletions including D564 resulted in constitutive receptor activity. Replacement of D564 by G, K, and N led to agonist-independent cAMP formation while introduction of a negatively charged E silenced constitutive receptor activity, indicating that an anionic amino acid at this position may be required to maintain an inactive receptor conformation. Insertion of A residues up- and downstream of D564 did not perturb receptor quiescence, showing that a certain degree of spatial freedom of the negatively charged amino acid within the context of the i3 loop is well tolerated. In contrast to the results obtained with the LH receptor, deletion of the corresponding D567 from the i3 loop of the FSH receptor did not cause constitutive receptor activation, highlighting significant differences in the activation mechanism of gonadotropin receptors.

Identification of cholinergic chemosensory cells in mouse tracheal and laryngeal glandular ducts.

Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect "bitter" substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel = TRPM5, phospholipase C(ß2)), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways.