The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H ؉ buffering polyamines by enhanced endosomal Cl ؊ accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl ؊ concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl ؊ ] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl ؊ or pH indicators. [Cl ؊ ] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl ؊ accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl ؊ accumulation and swelling/lysis were accounted for by the greater H ؉ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H ؉ /pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.Although gene delivery using non-viral vectors offers potential advantages over virus-based delivery systems, the relatively low transfection efficiency of non-viral vectors has been their major limitation for in vivo applications (1-4). The archetypal non-viral gene delivery system is the cationic polymer-DNA complex (polyplex), 1 in which plasmid DNA and a cationic carrier are condensed into a tight complex suitable for cellular internalization by endocytosis (5, 6). Transgene delivery to the nucleus is thought to require escape of the polyplex from endosomes, DNA/polymer dissociation, cytoplasmic DNA diffusion, and nuclear uptake (5, 7, 8). The low efficiency of polyplex escape from endosomes is thought to be an important determinant of the overall efficiency of non-viral gene transfer. Polyamines are useful polycationic macromolecules for nonviral gene transfer because of their high density of positive charges, ease of synthesis, and efficient polyplex formation (8 -10). Cationic polyamines with fixed, non-titratable charges such as polylysine (POL) are substantially less efficient at gene transfer than polyamines with titratable amines such as polyamidoamine (PAM) (11) and polyethylenimine (PEI) (12, 13). The lower efficiency is not caused by differences in morphology of the complexes or cell association (14). To explain this observation it has been postulated without direct evidence that the high H ϩ buffer capacity of polyamines containing titratable amines results in endosomal Cl Ϫ accumulation during acidification with presumed ...

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

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Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model, and near-complete suppression of cyst growth was found by tetrazolo-CFTR inh -172, a tetrazolo-derived thiazolidinone, and Ph-GlyH-101, a phenyl-derived glycine hydrazide, without an effect on cell proliferation. These compounds also inhibited cyst number and growth by Ͼ80% in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR inh -172 and Ph-GlyH-101 to neonatal, kidney-specific PKD1 knockout mice produced stable, therapeutic inhibitor concentrations of Ͼ3 M in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD.

Progressive acidification of vesicles in the endosomal pathway is important for receptor and ligand sorting and vesicular fusion (1, 2). Endosomal acidification is driven by a vacuolartype H ϩ pump that is present in the endosomal-limiting membrane. To maintain electroneutrality, H ϩ entry into the endosomal aqueous lumen must be accompanied by anion entry and/or cation exit. The principal transportable intracellular anion is Cl Ϫ and cation is K ϩ . Studies of organellar pH in living cells and isolated vesicles have provided evidence that Cl Ϫ entry may be the rate-limiting passive conductance in permitting active H ϩ entry in endosomes (3-9). In isolated endocytic vesicles from kidney proximal tubule (10) and liposomes reconstituted with proteins from clathrin-coated vesicles (11), a protein kinase A-activated Cl Ϫ conductance was characterized, and it was proposed that activation of Cl Ϫ channels might regulate endosomal acidification by providing a shunt to dissipate the interior-positive potential produce by the H ϩ pump. There is also evidence that Na ϩ /K ϩ pump activity in early endosomes may alter the driving force for H ϩ entry and thus regulate acidification (12-14). In Golgi, there is evidence that both Cl Ϫ and K ϩ conductances may contribute to acidification (15-17), whereas acidification of secretory granules in synaptic vesicles appears to require the expression of a specific Cl Ϫ channel (18). Although measurements of endosomal pH have been reported utilizing ratioable pH-sensitive fluorescent indicators (19 -22), there have been no measurements of ion concentrations in the endosomal lumen.Physico-chemical considerations indicate that endosomal [Cl Ϫ ] and pH should depend on the activity of the vacuolar H ϩ pump, the magnitude of endosomal cation (K ϩ , Na ϩ , and H ϩ ) and anion (Cl Ϫ and HCO 3 Ϫ ) conductances, endosomal membrane potential, buffer capacity, Donnan potential, and cytoplasmic pH and ion concentrations. Although attempts have been made to model endosomal/organelle acidification mathematically (23, 24), the paucity of information about key endosomal parameters precludes meaningful predictions about endosomal regulatory processes. Taken in reference to endosomal pH and cytoplasmic pH/[Cl Ϫ ], endosomal [Cl Ϫ ] is a particularly important parameter because of its implications for relative endosomal ion conductances and membrane potential. If Cl Ϫ conductance is the rate-limiting ion conductance in endosomal acidification, then the interior-positive endosomal electrical potential should produce marked Cl Ϫ accumulation in the endosomal aqueous lumen during acidification.The purpose of this study is to develop and apply methodology to measure endosomal [Cl Ϫ ] quantitatively in living cells. For these measurements, we synthesized a ratioable long wavelength fluorescent Cl Ϫ indicator that is brightly fluorescent, pH-insensitive, sensitive to [Cl Ϫ ] from 0 to Ͼ100 mM, biochemically stable, and membrane-impermeant. The endosomal aqueous lumen in cultured cells was stained with Cl Ϫ an...

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The ⌬F508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 M (with forskolin, 20 M) by an iodide uptake assay in epithelial cells coexpressing ⌬F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after ⌬F508-CFTR rescue by 24-h culture at 27°C. Secondary analysis and testing of Ͼ1000 structural analogs yielded two novel classes of correctors of defective ⌬F508-CFTR gating ("potentiators") with nanomolar potency that were active in human ⌬F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated ⌬F508-