ClC-5: role in endocytosis in the proximal tubule

The proper functioning of the Cl- channel, ClC-5, is essential for the uptake of low molecular mass proteins through receptor-mediated endocytosis in the proximal tubule. Dent's disease patients with mutant ClC-5 channels and ClC-5 knockout ( KO) mice both have low molecular mass proteinuria. To further understand the function of ClC-5, endocytosis was studied in LLC-PK1 cells and primary cultures of proximal tubule cells from wild-type (WT) and ClC-5 KO kidneys. Endocytosis in the proximal tubule cells from KO mice was reduced compared with that in WT animals. Endocytosis in WT but not in KO cells was inhibited by bafilomycin A-1 and Cl- depletion, whereas endocytosis in both WT and KO cells was inhibited by the NHE3 blocker, S3226. Infection with adenovirus containing WT ClC-5 rescued receptor-mediated endocytosis in KO cells, whereas infection with any of the three disease-causing mutants, myc-W22G-ClC5, myc-S520P-ClC-5, or myc-R704X-ClC-5, did not. WT and the three mutants all trafficked to the apical surface, as assessed by surface biotinylation. WT-ClC-5 and the W22G mutant were internalized similarly, whereas neither the S520P nor the R704X mutants was. These data indicate that ClC-5 is important for Cl- and proton pump-mediated endocytosis. However, not all receptor-mediated endocytosis in the proximal tubule is dependent on ClC-5. There is a significant fraction that can be inhibited by an NHE3 blocker. Our data from the mutants suggest that defective targeting and trafficking of mutant ClC-5 to the endosomes are a major determinant in the lack of normal endocytosis in Dent's disease

A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

Ca2+ activated Cl− channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl− secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca2+ and Cl− secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells

Syntaxin 8 and the Endoplasmic Reticulum Processing of ΔF508-CFTR

Background/Aims: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [1]. Methods: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508’s processing and movement out of the ER. Results: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control protein, Hsp 27, a protein that targets ΔF508-CFTR for sumoylation and subsequent degradation, to ΔF508-CFTR. STX8 silencing increased the levels of Hsp 60 a protein involving in early events in protein folding. Conclusion: STX8 knockdown creates an environment favorable for mature ΔF508 to reach the cell surface. The data also suggest that when present at normal levels, STX8 functions as part of the cell’s quality control mechanism

Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia

BACKGROUND: Adeno-associated virus type 2 (AAV-2) attachment and internalization is thought to be mediated by host cell membrane-associated heparan sulfate proteoglycans (HSPG). Lack of HSPG on the apical membrane of respiratory epithelial cells has been identified as a reason for inefficient rAAV-2 infection in pulmonary applications in-vivo. The aim of this investigation was to determine the necessity of cell membrane HSPG for efficient infection by rAAV-2. RESULTS: Rates of transduction with rAAV2-CMV-EGFP3 in several different immortalized airway epithelial cell lines were determined at different multiplicities of infection (MOI) before and after removal of membrane HSPG by heparinase III. Removal of HSPG decreased the efficacy of infection with rAAV2 by only 30–35% at MOI ≤ 100 for all of respiratory cell lines tested, and had even less effect at an MOI of 1000. Studies in mutant Chinese Hamster Ovary cell lines known to be completely deficient in surface HSPG also demonstrated only moderate effect of absence of HSPG on rAAV-2 infection efficacy. However, mutant CHO cells lacking all membrane proteoglycans demonstrated dramatic reduction in susceptibility to rAAV-2 infection, suggesting a role of membrane glycosaminoglycans other than HSPG in mediating rAAV-2 infection. CONCLUSION: Lack of cell membrane HSPG in pulmonary epithelia and other cell lines results in only moderate decrease in susceptibility to rAAV-2 infection, and this decrease may be less important at high MOIs. Other cell membrane glycosaminoglycans can play a role in permitting attachment and subsequent rAAV-2 internalization. Targeting alternative membrane glycosaminoglycans may aid in improving the efficacy of rAAV-2 for pulmonary applications

Membrane topology of aquaporin CHIP. Analysis of functional epitope-scanning mutants by vectorial proteolysis

CHIP is the archetypal member of the aquaporins, a widely expressed family of membrane water channels. The NH2- and COOH-terminal halves of CHIP are sequence-related, and hydropathy analysis predicted six membrane-spanning domains with five connecting loops (A-E). Here, we determined the membrane topology of CHIP expressed in Xenopus oocytes using biologically active recombinant channels. CHIP is glycosylated at Asn-42, indicating loop A is exofacial. An epitope from the coronavirus E1 glycoprotein was inserted into CHIP and localized to the outer or inner leaflet of the membrane by alpha-chymotrypsin digestion of intact oocytes or inside-out membrane vesicles. The E1 epitope at Thr-120 was protease-sensitive in intact oocytes, indicating that loop C is exofacial. The E1 epitope at Lys-6, Arg-162, or Lys-267 was protease-sensitive in inside-out membrane vesicles, confirming the cytoplasmic location of the NH2 and COOH termini and loop D. Insertions into loops B and E did not produce active water channels, but their cleavage patterns were consistent with inner (loop B) and outer (loop E) leaflet locations. This study indicates that the functional CHIP molecule is a unique structure with two internal repeats oriented 180 degrees to each other within the membrane

The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel

Water channels provide the plasma membranes of red cells and renal proximal tubules with high permeability to water, thereby permitting water to move in the direction of an osmotic gradient. Molecular identification of CHIP28 protein as the membrane water channel was first accomplished by measurement of osmotic swelling of Xenopus oocytes injected with CHIP28 RNA (Preston, G.M., Carroll, T.P., Guggino, W.B., and Agre, P. (1992) Science 256, 385-387). Since water channels are pharmacologically inhibited by submillimolar concentrations of Hg2+, site-directed mutagenesis was undertaken to demonstrate which of the 4 cysteines (87, 102, 152, or 189) is the Hg(2+)-sensitive residue in the CHIP28 molecule. Each cysteine was individually replaced by serine, and oocytes expressing each of the four mutants exhibited osmotic water permeability (Pf) equivalent to wild-type CHIP28. After incubation in HgCl2, all were significantly inhibited, except C189S exists as a multisubunit complex in the native membrane; however, although oocytes injected with mixed CHIP28 and C189S RNAs exhibited Pf corresponding to the sum of their individual activities, exposure to Hg2+ only reduced the Pf to the level of the C189S mutant. Of the six substitutions at residue 189, only the serine and alanine mutants exhibited increased Pf and had glycosylation patterns resembling wild-type CHIP28 on immunoblots. These studies demonstrated: (i) CHIP28 water channel activity is retained despite substitution of individual cysteines with serine; (ii) cysteine 189 is the Hg(2+)-sensitive residue; (iii) the subunits of the CHIP28 complex are individually active water pores; (iv) residue 189 is critical to proper processing of the CHIP28 protein

Characterization of aquaporin-6 as a nitrate channel in mammilian cells: Requirement of pore-lining residue threonine 63

Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg2+ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH-induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl- revealed high nitrate permeability even at pH 7.4; P(NO3)/P(Cl) > 9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO3-/Cl- selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO3-/Cl- mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport

A regulatory role of polycystin-1 on cystic fibrosis transmembrane conductance regulator plasma membrane expression.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by genetic mutations in either PKD1 or PKD2, the genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. ADPKD is characterized by the formation of multiple, progressive, fluid-filled renal cysts. To elucidate the mechanism of fluid secretion by ADPKD cysts, we examined the effect of PC-1 on the plasma membrane expression of cystic fibrosis transmembrane conductance regulator (CFTR), a key Cl(-) secretory protein. Five stably transfected MDCK lines were used in this study: two transfected with empty vector (control cells) and three expressing human PC-1 (PC-1 cells). The cAMP-induced endogenous short circuit currents (I(sc)) were smaller in PC-1 cells than in control cells. Compared to control cells, PC-1 cells transiently expressing pEGFP-CFTR showed significant reduction of whole cell cAMP-activated Cl(-) currents. Cell surface biotinylation experiments also indicated a reduction in surface expression of CFTR in PC-1 cells compared to control. Furthermore, studies using CHO cells transiently expressing PC-1 and CFTR suggest the importance of the PC-1 COOH-terminus in the observed reduction of CFTR plasma membrane expression. No differences in either endogeneous K(+) currents or P2Y receptor responses were observed between PC-1 and control cells, indicating the specificity of PC-1's action. These results indicate that PC-1 selectively maintains low cell surface expression of CFTR. Moreover, these findings suggest that the malfunction of PC-1 enhances plasma membrane expression of CFTR, thus causing abnormal Cl(-)secretion into the cyst lumen

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule