Bryan D. Moyer

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl−) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl− secretion by stimulating CFTR Cl− channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl− secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl− secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells.

Sodium butyrate and its derivatives are useful therapeutic agents for the treatment of genetic diseases including urea cycle disorders, sickle cell disease, thalassemias, and possibly cystic fibrosis (CF). Butyrate partially restores cAMP-activated Cl(-) secretion in CF epithelial cells by stimulating DeltaF508 cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) gene expression and increasing the amount of DeltaF508-CFTR in the plasma membrane. Because the effect of butyrate on Cl(-) secretion by renal epithelial cells has not been reported, we examined the effects of chronic butyrate treatment (15-18 h) on the function, expression, and localization of CFTR fused to the green fluorescent protein (GFP-CFTR) in stably transfected MDCK cells. We report that sodium butyrate reduced Cl(-) secretion across MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold and increased apical membrane Cl(-) currents 30-fold. Although butyrate also increased Na-K-ATPase protein expression twofold, the drug reduced the activity of the Na-K-ATPase by 55%. Our findings suggest that butyrate inhibits cAMP-stimulated Cl(-) secretion across MDCK cells in part by reducing the activity of the Na-K-ATPase.

Small Molecule Activator of the Human Epithelial Sodium Channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of α, β, and γ subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na+ entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the β subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of αβG37Sγ hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na+ flux across epithelial membranes is clinically desirable.

PBA increases CFTR expression but at high doses inhibits Cl− secretion in Calu-3 airway epithelial cells

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl(-) secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl(-) secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na(+)-K(+)-ATPase. PBA reduced CFTR Cl(-) currents across the apical membrane but had no effect on Na(+)-K(+)-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl(-) secretion by inhibiting CFTR Cl(-) currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05-2 mM) had no effect on cAMP-stimulated Cl(-) secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl(-) secretion. However, concentrations >5 mM might reduce transepithelial Cl(-) secretion by serous cells in submucosal glands in individuals expressing wt CFTR.