The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br >> NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.

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The epithelial sodium channel (ENaC) is regulated by epidermal growth factor (EGF). We investigate whether ENaC is regulated by another EGF receptor (EGFR) ligand, transforming growth factor-alpha (TGF-alpha). We show that chronic (24 h) treatment with TGF-alpha inhibits ENaC in Xenopus laevis kidney cells 20 times more strongly than EGF. By using single-channel measurements, we show that TGF-alpha significantly reduces the number of ENaC per patch. The open probability (P(o)) is unchanged by 24-h treatment with TGF-alpha. alpha-, beta-, and gamma-ENaC mRNA levels are significantly reduced by TGF-alpha or EGF. TGF-alpha or EGF reduces alpha- and gamma-ENaC proteins in the membrane; however, beta-ENaC is unchanged. TGF-alpha or EGF inhibits ENaC by activating EGFR since the EGFR inhibitor AG1478 blocks the effects of both. The MAPK 1/2 inhibitor U0126 also blocks the effect of TGF-alpha or EGF on ENaC, indicating that the MAPK1/2 pathway is involved in the TGF-alpha- or EGF-induced inhibition of ENaC. Interestingly, acute treatment (<1 h) with TGF-alpha or EGF does not inhibit ENaC current; it enhances ENaC activity by increasing P(o). Pretreatment of the cells with U0126 potentiates the acute TGF-alpha- or EGF-induced stimulation of ENaC. This TGF-alpha- or EGF-induced increase in sodium current is abolished by a phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, LY294002, suggesting that PI-3 kinase is involved in the activation of sodium transport. In conclusion, chronic treatment with TGF-alpha or EGF inhibits ENaC by decreasing the number of channels in the membrane transcriptionally through MAPK1/2 pathways, but acute treatment with TGF-alpha or EGF activates ENaC by increasing P(o) via PI-3 kinase.

Female sex predisposes individuals to poorer outcomes during respiratory disorders like cystic fibrosis and influenza-associated pneumonia. A common link between these disorders is dysregulation of alveolar fluid clearance via disruption of epithelial sodium channel (ENaC) activity. Recent evidence suggests that female sex hormones directly regulate expression and activity of alveolar ENaC. In our study, we identified the mechanism by which estradiol (E2) or progesterone (P4) independently regulates alveolar ENaC. Using cell-attached patch clamp, we measured ENaC single-channel activity in a rat alveolar cell line (L2) in response to overnight exposure to either E2 or P4. In contrast to P4, E2 increased ENaC channel activity (NPo) through an increase in channel open probability (Po) and an increased number of patches with observable channel activity. Apical plasma membrane abundance of the ENaC α-subunit (αENaC) more than doubled in response to E2 as determined by cell surface biotinylation. αENaC membrane abundance was approximately threefold greater in lungs from female rats in proestrus, when serum E2 is greatest, compared with diestrus, when it is lowest. Our results also revealed a significant role for the G protein-coupled estrogen receptor (Gper) to mediate E2's effects on ENaC. Overall, our results demonstrate that E2 signaling through Gper selectively activates alveolar ENaC through an effect on channel gating and channel density, the latter via greater trafficking of channels to the plasma membrane. The results presented herein implicate E2-mediated regulation of alveolar sodium channels in the sex differences observed in the pathogenesis of several pulmonary diseases.