Cl- as a bona fide signaling ion.

Cl- is the major extracellular (Cl-out) and intracellular (Cl-in) anion whose concentration is actively regulated by multiple transporters. These transporters generate Cl- gradients across the plasma membrane and between the cytoplasm and intracellular organelles. [Cl-]in changes rapidly in response to cell stimulation and influences many physiological functions, as well as cellular and systemic homeostasis. However, less appreciated is the signaling function of Cl-. Cl- interacts with multiple proteins to directly modify their activity. This review highlights the signaling function of Cl- and argues that Cl- is a bona fide signaling ion, a function deserving extensive exploration.

Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca2+ signaling complexes at the ER/PM compartment.

Communication and material transfer between membranes and organelles take place at membrane contact sites (MCSs). MCSs between the ER and PM, the ER/PM junctions, are the sites where the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1 cluster. MCSs are formed by tether proteins that bridge the opposing membranes, but the identity and role of these tethers in receptor-evoked Ca2+ signaling is not well understood. Here, we identified Anoctamin 8 (ANO8) as a key tether in the formation of the ER/PM junctions that is essential for STIM1-STIM1 interaction and STIM1-Orai1 interaction and channel activation at a ER/PM PI(4,5)P2-rich compartment. Moreover, ANO8 assembles all core Ca2+ signaling proteins: Orai1, PMCA, STIM1, IP3 receptors, and SERCA2 at the ER/PM junctions to mediate a novel form of Orai1 channel inactivation by markedly facilitating SERCA2-mediated Ca2+ influx into the ER. This controls the efficiency of receptor-stimulated Ca2+ signaling, Ca2+ oscillations, and duration of Orai1 activity to prevent Ca2+ toxicity. These findings reveal the central role of MCSs in determining efficiency and fidelity of cell signaling.

Modulation of Cl- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl- in Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl- in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl- in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3 - secretion.

A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells.

TRPC6 plays important human physiological functions, notably in artery and arterioles constriction, in regulation of vascular volume and in bronchial muscle constriction. It is implicated in pulmonary hypertension, cardiovascular disease, and focal segmental glomerulosclerosis and seems to play a role in cancer development. Previously, we identified Guanabenz, an α2-adrenergic agonist used for hypertension treatment (Wytensin®), as an activator of calcium-dependent chloride channels (CaCC) in human Cystic Fibrosis (CF) nasal epithelial cells by transiently increasing [Ca2+]i via an influx of extracellular Ca2+. In this study, using assays to measure chloride channel activity, we show that guanabenz is an activator of CaCC in freshly dissociated human bronchial epithelial cells from three CF patients with various genotypes (F508del/F508del, F508del/R1066C, F508del/H1085R). We further characterised the effect of guanabenz and show that it is independent of α-adrenergic receptors, is inhibited by the TRPC family inhibitor SKF-96365 but not by the TRPV family inhibitor ruthenium red. Using western-blotting, Ca2+ measurements and iodide efflux assay, we found that TRPC1 siRNA has no effect on guanabenz induced responses whereas TRPC6 siRNA prevented the guanabenz-dependent Ca2+ influx and the CaCC-dependent activity stimulated by guanabenz. In conclusion, we show that TRPC6 channel is pivotal for the activation of CaCC by guanabenz through a α2-adrenergic-independent pathway in human airway epithelial cells. We suggest propose a functional coupling between TRPC6 and CaCC and guanabenz as a potential TRPC6 activator for exploring TRPC6 and CaCC channel functions and corresponding channelopathies.

The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity.

Increase of Ca(2+) influx in Cystic Fibrosis (CF) cells has been reported to be related to Transient Receptor Potential Canonical (TRPC6) channel, which is implicated in a functional coupling with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Several members of the Transient Receptor Potential Vanilloid (TRPV) channels family have already been described as emerging target for respiratory diseases. Two specific isoforms, TRPV5 and TRPV6 are of particular interest in the context of CF Ca(2+) homeostasis as they are highly selective toward Ca(2+) and constitutively activated. Thus, we investigated the involvement of these channels in Ca(2+) influx in CF and non-CF human bronchial epithelial cell lines. 16HBE14o-, CFBE41o- cell lines, primary human airway epithelial cells (hAEC) and freshly isolated human airway epithelial cells from CF and non-CF individuals were used. We showed that both channels are expressed in CF and non-CF cells and constitutive Ca(2+) influx was significantly higher (85%) in cells from CF individuals compared to cells from non-CF ones. Using the selective inhibitor of TRPV6 channel SOR-C27 and a siRNA strategy, our results revealed that TRPV6 was mostly involved in the increase of Ca(2+) influx. TRPV6 channel is negatively regulated by the PLC-PIP2 pathway. We measured the Ca(2+) influx in the presence of the non-specific PLC inhibitor, U73122, in non-CF human bronchial epithelial cells. Ca(2+) influx was increased by 33% with U73122 and this increase was largely reduced in the presence of SOR-C27. PLC inhibition in CF cells by U73122 had no effect on Ca(2+) influx. These results showed that PLC-PIP2 pathway is dysregulated in CF cells and leads to the increase of TRPV6 activity. The regulation of TRPV6 by PLC-PIP2 pathway implicates the specific PLC isoform, PLC-δ1. Immunoblot experiments revealed that expression of PLC-δ1 was decreased by 70% in CF cells. TRPV6 activity was normalized but not the level of expression of PLC-δ1 protein after F508del-CFTR rescue by low temperature for 48 h or treated for 24 h by 10 µM VX-809 in CF cells. This study revealed TRPV6 and PLC-δ1 as critical actor of Ca(2+) homeostasis in CF human bronchial epithelial cells.

Effect of VX-770 (ivacaftor) and OAG on Ca2+ influx and CFTR activity in G551D and F508del-CFTR expressing cells.

BACKGROUND: TRPC6 has been proposed to be responsible for the abnormal OAG-dependent Ca(2+) influx in cystic fibrosis (CF) cells and we hypothesized that it interacts with CFTR. Here, we investigated how this functional complex operates in CF and non-CF epithelial cells. METHODS: Chinese hamster ovary (CHO) cells stably transfected with pNut vector containing wild type CFTR (CHO-WT), F508del-CFTR (CHO-F508del) or G551D-CFTR(CHO-G551D) were used. Calcium channel activity was recorded using Fluo-4 probe and CFTR activity was measured by iodide efflux technique in the presence of CFTR activators (forskolin, genistein) and VX-770, CFTR inhibitor (GPinh5a) and TRPC non-selective modulators (OAG, SKF96365). RESULTS: CFTR down regulates OAG Ca(2+) response and OAG Ca(2+) influx increases CFTR chloride efflux. Furthermore, we observed potentiation of G551D-CFTR activity when combining VX-770 and OAG. CONCLUSION: Taking advantage of the functional coupling between OAG-dependent Ca(2+) influx and CFTR, a combination of OAG and VX-770 could be a therapeutic strategy for homozygote patients bearing the G551D-CFTR mutation.