A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Structural view of G protein-coupled receptor signaling in the retinal rod outer segment.

Visual phototransduction is the most extensively studied G protein-coupled receptor (GPCR) signaling pathway because of its quantifiable stimulus, non-redundancy of genes, and immense importance in vision. We summarize recent discoveries that have advanced our understanding of rod outer segment (ROS) morphology and the pathological basis of retinal diseases. We have combined recently published cryo-electron tomography (cryo-ET) data on the ROS with structural knowledge on individual proteins to define the precise spatial limitations under which phototransduction occurs. Although hypothetical, the reconstruction of the rod phototransduction system highlights the potential roles of phosphodiesterase 6 (PDE6) and guanylate cyclases (GCs) in maintaining the spacing between ROS discs, suggesting a plausible mechanism by which intrinsic optical signals are generated in the retina.

Gßγ activates PIP2 hydrolysis by recruiting and orienting PLCß on the membrane surface.

Phospholipase C-ßs (PLCßs) catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text]  [Formula: see text]. [Formula: see text] regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca2+ levels and activate protein kinase C, respectively. PLCßs are regulated by G protein-coupled receptors through direct interaction with [Formula: see text] and [Formula: see text] and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which [Formula: see text] activates PLCß3. We show that PLCß3 functions as a slow Michaelis-Menten enzyme ( [Formula: see text] ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of PLCß3. Its partition coefficient is such that only a small quantity of PLCß3 exists in the membrane in the absence of [Formula: see text] . When [Formula: see text] is present, equilibrium binding on the membrane surface increases PLCß3 in the membrane, increasing [Formula: see text] in proportion. Atomic structures on membrane vesicle surfaces show that two [Formula: see text] anchor PLCß3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that [Formula: see text] activates PLCß by increasing its concentration on the membrane surface and orienting its catalytic core to engage [Formula: see text] . This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by [Formula: see text], IP3, and DAG.

The mechanism of Gαq regulation of PLCß3-catalyzed PIP2 hydrolysis.

PLCß (Phospholipase Cß) enzymes cleave phosphatidylinositol 4,5-bisphosphate (PIP2) producing IP3 and DAG (diacylglycerol). PIP2 modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca2+ levels and protein phosphorylation by protein kinase C, respectively. PLCß enzymes are under the control of G protein coupled receptor signaling through direct interactions with G proteins Gßγ and Gαq and have been shown to be coincidence detectors for dual stimulation of Gαq and Gαi-coupled receptors. PLCßs are aqueous-soluble cytoplasmic enzymes but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that Gßγ activates PLCß3 by recruiting it to the membrane. Using these same methods, here we show that Gαq increases the catalytic rate constant, kcat, of PLCß3. Since stimulation of PLCß3 by Gαq depends on an autoinhibitory element (the X-Y linker), we propose that Gαq produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the PLCß3·Gαq and PLCß3·Gßγ(2)·Gαq complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate PLCß3 activity. The structures rationalize a finding in the enzyme assay, that costimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of PLCß3 is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.

Coexistence within one cell of microvillous and ciliary phototransductions across M1- through M6-IpRGCs.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) serve as primary photoceptors by expressing the photopigment, melanopsin, and also as retinal relay neurons for rod and cone signals en route to the brain, in both cases for the purpose of non-image vision as well as aspects of image vision. So far, six subtypes of ipRGCs (M1 through M6) have been characterized. Regarding their phototransduction mechanisms, we have previously found that, unconventionally, rhabdomeric (microvillous) and ciliary signaling motifs co-exist within a given M1-, M2-, and M4-ipRGC, with the first mechanism involving PLCß4 and TRPC6,7 channels and the second involving cAMP and HCN channels. We have now examined M3-, M5-, and M6-cells and found that each cell likewise uses both signaling pathways for phototransduction, despite differences in the percentage representation by each pathway in a given ipRGC subtype for bright-flash responses (and saturated except for M6-cells). Generally, M3- and M5-cells show responses quite similar in kinetics to M2-responses, and M6-cell responses resemble broadly those of M1-cells although much lower in absolute sensitivity and amplitude. Therefore, similar to rod and cone subtypes in image vision, ipRGC subtypes possess the same phototransduction mechanism(s) even though they do not show microvilli or cilia morphologically.

Structural insights into galanin receptor signaling.

Galanin is a biologically active neuropeptide, and functions through three distinct G protein­coupled receptors (GPCRs), namely GALR1, GALR2, and GALR3. GALR signaling plays important roles in regulating various physiological processes such as energy metabolism, neuropathic pain, epileptic activity, and sleep homeostasis. GALR1 and GALR3 signal through the Gi/o pathway, whereas GALR2 signals mainly through the Gq/11 pathway. However, the molecular basis for galanin recognition and G protein selectivity of GALRs remains poorly understood. Here, we report the cryoelectron microscopy structures of the GALR1-Go and the GALR2-Gq complexes bound to the endogenous ligand galanin or spexin. The galanin peptide mainly adopts an alpha helical structure, which binds at the extracellular vestibule of the receptors, nearly parallel to the membrane plane without penetrating deeply into the receptor core. Structural analysis combined with functional studies reveals important structural determinants for the G protein selectivity of GALRs as well as other class A GPCRs. In addition, we show that the zinc ion is a negative allosteric regulator of GALR1 but not GALR2. Our studies provide insight into the mechanisms of G protein selectivity of GPCRs and highlight a potential function of the neuromodulator zinc ion as a modulator of GPCR signaling in the central nervous system.

Low signaling efficiency from receptor to effector in olfactory transduction: A quantified ligand-triggered GPCR pathway.

G protein-coupled receptor (GPCR) signaling is ubiquitous. As an archetype of this signaling motif, rod phototransduction has provided many fundamental, quantitative details, including a dogma that one active GPCR molecule activates a substantial number of downstream G protein/enzyme effector complexes. However, rod phototransduction is light-activated, whereas GPCR pathways are predominantly ligand-activated. Here, we report a detailed study of the ligand-triggered GPCR pathway in mammalian olfactory transduction, finding that an odorant-receptor molecule when (one-time) complexed with its most effective odorants produces on average much less than one downstream effector. Further experiments gave a nominal success probability of tentatively ∼10-4 (more conservatively, ∼10-2 to ∼10-5). This picture is potentially more generally representative of GPCR signaling than is rod phototransduction, constituting a paradigm shift.

GPCRs and fibroblast heterogeneity in fibroblast-associated diseases.

G protein-coupled receptors (GPCRs) are the largest and most diverse class of signaling receptors. GPCRs regulate many functions in the human body and have earned the title of "most targeted receptors". About one-third of the commercially available drugs for various diseases target the GPCRs. Fibroblasts lay the architectural skeleton of the body, and play a key role in supporting the growth, maintenance, and repair of almost all tissues by responding to the cellular cues via diverse and intricate GPCR signaling pathways. This review discusses the dynamic architecture of the GPCRs and their intertwined signaling in pathological conditions such as idiopathic pulmonary fibrosis, cardiac fibrosis, pancreatic fibrosis, hepatic fibrosis, and cancer as opposed to the GPCR signaling of fibroblasts in physiological conditions. Understanding the dynamics of GPCR signaling in fibroblasts with disease progression can help in the recognition of the complex interplay of different GPCR subtypes in fibroblast-mediated diseases. This review highlights the importance of designing and adaptation of next-generation strategies such as GPCR-omics, focused target identification, polypharmacology, and effective personalized medicine approaches to achieve better therapeutic outcomes for fibrosis and fibrosis associated malignancies.

G Protein-Coupled Receptor Dimerization-What Next?

Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.

A negative feedback loop in the GPCR pathway underlies efficient coding of external stimuli.

Efficient navigation based on chemical cues is an essential feature shared by all animals. These cues may be encountered in complex spatiotemporal patterns and with orders of magnitude varying intensities. Nevertheless, sensory neurons accurately extract the relevant information from such perplexing signals. Here, we show how a single sensory neuron in Caenorhabditis elegans animals can cell-autonomously encode complex stimulus patterns composed of instantaneous sharp changes and of slowly changing continuous gradients. This encoding relies on a simple negative feedback in the G-protein-coupled receptor (GPCR) signaling pathway in which TAX-6/Calcineurin plays a key role in mediating the feedback inhibition. This negative feedback supports several important coding features that underlie an efficient navigation strategy, including exact adaptation and adaptation to the magnitude of the gradient's first derivative. A simple mathematical model explains the fine neural dynamics of both wild-type and tax-6 mutant animals, further highlighting how the calcium-dependent activity of TAX-6/Calcineurin dictates GPCR inhibition and response dynamics. As GPCRs are ubiquitously expressed in all sensory neurons, this mechanism may be a general solution for efficient cell-autonomous coding of external stimuli.

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration.

BACKGROUND: G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer. METHODS: We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, ß-arrestin recruitment, ligand binding, and transwell migration assays. RESULTS: We observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and ß-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists. CONCLUSIONS: The presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions. Video Abstract.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.

Deciphering the signaling mechanisms of ß-arrestin1 and ß-arrestin2 in regulation of cancer cell cycle and metastasis.

ß-Arrestins are ubiquitously expressed intracellular proteins with many functions which interact directly and indirectly with a wide number of cellular partners and mediate downstream signaling. Originally, ß-arrestins were identified for their contribution to GPCR desensitization to agonist-mediated activation, followed by receptor endocytosis and ubiquitylation. However, current investigations have now recognized that in addition to GPCR arresting (hence the name arrestin). ß-Arrestins are adaptor proteins that control the recruitment, activation, and scaffolding of numerous cytoplasmic signaling complexes and assist in G-protein receptor signaling, thus bringing them into close proximity. They have participated in various cellular processes such as cell proliferation, migration, apoptosis, and transcription via canonical and noncanonical pathways. Despite their significant recognition in several physiological processes, these activities are also involved in the onset and progression of various cancers. This review delivers a concise overview of the role of ß-arrestins with a primary emphasis on the signaling processes which underlie the mechanism of ß-arrestins in the onset of cancer. Understanding these processes has important implications for understanding the therapeutic intervention and treatment of cancer in the future.

N-Acyl Amides from Neisseria meningitidis and Their Role in Sphingosine Receptor Signaling.

Neisseria meningitidis is a Gram-negative opportunistic pathogen that is responsible for causing human diseases with high mortality, such as septicemia and meningitis. The molecular mechanisms N. meningitidis employ to manipulate the immune system, translocate the mucosal and blood-brain barriers, and exert virulence are largely unknown. Human-associated bacteria encode a variety of bioactive small molecules with growing evidence for N-acyl amides as being important signaling molecules. However, only a small fraction of these metabolites has been identified from the human microbiota thus far. Here, we heterologously expressed an N-acyltransferase encoded in the obligate human pathogen N. meningitidis and identified 30 N-acyl amides with representative members serving as agonists of the G-protein coupled receptor (GPCR) S1PR4. During this process, we also characterized two mammalian N-acyl amides derived from the bovine medium. Both groups of metabolites suppress anti-inflammatory interleukin-10 signaling in human macrophage cell types, but they also suppress the pro-inflammatory interleukin-17A+ population in TH 17-differentiated CD4+ T cells.

QR code model: a new possibility for GPCR phosphorylation recognition.

G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in the human body and are responsible for accurately transmitting extracellular information to cells. Arrestin is an important member of the GPCR signaling pathway. The main function of arrestin is to assist receptor desensitization, endocytosis and signal transduction. In these processes, the recognition and binding of arrestin to phosphorylated GPCRs is fundamental. However, the mechanism by which arrestin recognizes phosphorylated GPCRs is not fully understood. The GPCR phosphorylation recognition "bar code model" and "flute" model describe the basic process of receptor phosphorylation recognition in terms of receptor phosphorylation sites, arrestin structural changes and downstream signaling. These two models suggest that GPCR phosphorylation recognition is a process involving multiple factors. This process can be described by a "QR code" model in which ligands, GPCRs, G protein-coupled receptor kinase, arrestin, and phosphorylation sites work together to determine the biological functions of phosphorylated receptors. Video Abstract.

Ca2+ allostery in PTH-receptor signaling.

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3',5'-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein-coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.

The Role of Reversible Phosphorylation of Drosophila Rhodopsin.

Vertebrate and fly rhodopsins are prototypical GPCRs that have served for a long time as model systems for understanding GPCR signaling. Although all rhodopsins seem to become phosphorylated at their C-terminal region following activation by light, the role of this phosphorylation is not uniform. Two major functions of rhodopsin phosphorylation have been described: (1) inactivation of the activated rhodopsin either directly or by facilitating binding of arrestins in order to shut down the visual signaling cascade and thus eventually enabling a high-temporal resolution of the visual system. (2) Facilitating endocytosis of activated receptors via arrestin binding that in turn recruits clathrin to the membrane for clathrin-mediated endocytosis. In vertebrate rhodopsins the shutdown of the signaling cascade may be the main function of rhodopsin phosphorylation, as phosphorylation alone already quenches transducin activation and, in addition, strongly enhances arrestin binding. In the Drosophila visual system rhodopsin phosphorylation is not needed for receptor inactivation. Its role here may rather lie in the recruitment of arrestin 1 and subsequent endocytosis of the activated receptor. In this review, we summarize investigations of fly rhodopsin phosphorylation spanning four decades and contextualize them with regard to the most recent insights from vertebrate phosphorylation barcode theory.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects.

The interplay between G protein-coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain.

Phospholipase C controls chloride-dependent short-circuit current in human bronchial epithelial cells.

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here, we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over-expressing wild type (WT)- or F508del-CFTR. Western blot analysis shows expression of the three endogenous phospholipase C (PLC) isoforms, namely, PLCδ1, PLCγ1, and PLCß3 in 16HBE14o- cells. In 16HBE14o- cells, we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCß3 and PLCγ1 in CFTR-dependent short-circuit current activated by forskolin, but not of PLCδ1. In CFBE-WT CFTR and corrected CFBE-F508del CFTR cells, PLCß3 silencing also inhibits CFTR-dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR-dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells.