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ClC-4 is closely related to ClC-5, a member of the ClC family of transporters and channels. Unlike ClC-5, for which a role in the regulation of endosomal function was well established, the cellular function of ClC-4 was uncertain. In the present study, we tested for a specific role for ClC-4 in recycling endosomes by comparing transferrin (Tfn) receptor function in primary cell lines generated from ClC-4-null mice and their wild-type siblings. We found that endosomal pH is relatively alkaline and receptor-mediated uptake of Tfn is reduced in ClC-4-null fibroblasts. Surprisingly, this reduction in Tfn uptake occurs, despite a minor increase in the total surface expression of the Tfn receptor in ClC-4-null fibroblasts. As impaired Tfn uptake by ClC-4-null fibroblasts could be rescued to wild-type levels by addition of the iron chelator: desoxiferramine, the primary defect in these cells is related to the failure of iron to dissociate from Tfn, a pH-dependent event in endosomes that precedes the dissociation of Tfn from its receptor at the cell surface. Interestingly, ClC-4 depletion had no effect on epidermal growth factor receptor (EGFR) trafficking to lysosomes for degradation pointing to its specific role in recycling endosomes. These observations provide direct evidence supporting an essential role for ClC-4 in the modulation of Tfn receptor accessibility at the cell surface through its role in endosomal acidification.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.

It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.

Mutations in the KvLQT1 gene are the cause of the long QT syndrome 1. KvLQT1 gene product is associated with the regulator protein IsK to produce a component of the delayed rectifier K+ current in cardiac myocytes. We identified an N-terminal truncated isoform of the KvLQT1 gene product, referred to as isoform 2. In RNase protection assays, isoform 2 represented 28.1 +/- 0.6% of the total KvLQT1 expression in the human adult ventricle. COS-7 cells injected intranuclearly with KvLQT1 isoform 1 cDNA exhibited a fast-activating K+ current, whereas those injected with a KvLQT1 isoform 1 plus IsK cDNA showed a slow-activating K+ current. Cells injected with KvLQT1 isoform 2 plasmid showed no detectable K+ current. Those injected with a 1/1 isoform 2/isoform 1 ratio showed no detectable K+ current. Those injected with 1/5 and 2/5 ratios showed a K+ current with markedly reduced amplitude. Coexpression of the IsK regulator consistently reduced the dominant negative effects of isoform 2. Our results indicate that KvLQT1 isoform 2 exerts a pronounced negative dominance on isoform 1 channels and that the cardiac KvLQT1 K+ channel complex is composed of at least three different proteins as follows: isoform 1, isoform 2, and IsK.

The perforated-patch configuration of the patch-clamp technique was used to record whole cell currents from human epithelial CFPAC-1 cells defective for functional cystic fibrosis transmembrane conductance regulator (CFTR). In CFPAC-1 cells, adenosine 3',5'-cyclic monophosphate (cAMP) stimulation with forskolin (10 microM) plus 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) activated neither Cl- nor K+ currents. In the same cells transfected with wild-type CFTR gene, cAMP stimulation produced activation of both Cl- and K+ currents. In Cl(-)-depleted medium (gluconate as a substitute), cAMP stimulation evoked a K+ current in CFTR-transfected but not in untransfected CFPAC-1 cells. This cAMP-evoked K+ current was the sum of two components: 1) a time-independent inwardly rectifying component, and 2) a slowly relaxing component activated at positive voltages. Increasing intracellular Ca2+ with ionomycin (1 microM) activated K+ currents in either transfected or untransfected cells. In transfected cells, blocking the CFTR conductance with high-concentration glibenclamide (100 microM) reduced the K+ current when activated by cAMP but not when activated by Ca2+. Pretreating CFTR-transfected cells for 48 h with interferon-gamma downregulated CFTR gene expression and reduced cAMP but not Ca2+ activation of the whole cell K+ current. From these results, we conclude that functional membrane CFTR protein influences activation by cAMP of epithelial K+ currents.

Mutations in the gene coding for the chloride channel ClC-5 cause Dent's disease, a disease associated with proteinuria and renal stones. Studies in ClC-5 knockout mice suggest that this phenotype is related to defective endocytosis of low molecular weight proteins and membrane proteins by the renal proximal tubule. In this study, confocal micrographs of proximal tubules and cultured epithelial cells revealed that the related protein ClC-4 is expressed in endosomal membranes suggesting that this channel may also contribute to the function of this organelle. In support of this hypothesis, specific disruption of endogenous ClC-4 expression by transfection of ClC-4 antisense cDNA acidified endosomal pH and altered transferrin trafficking in cultured epithelial cells to the same extent as the specific disruption of ClC-5. Both channels can be co-immunoprecipitated, arguing that they may partially contribute to endosomal function as a channel complex. These studies prompt future investigation of the role of ClC-4 in renal function in health and in Dent's disease. Future studies will assess whether the severity of Dent's disease relates not only to the impact of particular mutations on ClC-5 but also on the consequences of those mutations on the functional expression of ClC-4.

The ClC-2 chloride channel has been implicated in essential physiological functions. Analyses of ClC-2 knock-out mice suggest that ClC-2 expression in retinal pigment epithelia and Sertoli cells normally supports the viability of photoreceptor cells and male germ cells, respectively. Further, other studies suggest that ClC-2 expression in neurons may modify inhibitory synaptic transmission via the gamma-aminobutyric acid, type A receptor. However, complete understanding of the physiological functions of ClC-2 requires elucidation of the molecular basis for its regulation. Using cell imaging and biochemical and electrophysiological techniques, we show that expression of ClC-2 at the cell surface may be regulated via an interaction with the dynein motor complex. Mass spectrometry and Western blot analysis of eluate from a ClC-2 affinity matrix showed that heavy and intermediate chains of dynein bind ClC-2 in vitro. The dynein intermediate chain co-immunoprecipitates with ClC-2 from hippocampal membranes suggesting that they also interact in vivo. Disruption of dynein motor function perturbs ClC-2 localization and increases the functional expression of ClC-2 in the plasma membranes of COS7 cells. Thus, cell surface expression of ClC-2 may be regulated by dynein motor activity. This work is the first to demonstrate an in vivo interaction between an ion channel and the dynein motor complex.

Biophysical properties of ROMK2 channel were investigated at physiological temperature, after reexpression of the recombinant ROMK2 protein in a mammalian cell expression system (COS-7). We observed that ROMK2 induced an inwardly rectifying K+ current whether polyvalent cations were present or not. Above +10 mV, ROMK2-induced current exhibited a voltage- and time-dependent decay, consistent with an inactivation process. Inactivation of ROMK2-induced current was also seen in inside out patch from ROMK2-expressing Xenopus oocyte. In COS-7 cells, inactivation was found to account for most of the inward rectification. Mg2+ and spermine modulated rectification by accelerating inactivation kinetics independently of membrane potential. These results establish for the first time ROMK2 properties in a mammalian cell expression system.

In cardiac myocytes, the slow component of the delayed rectifier K+current ( IKs) is regulated by cAMP. Elevated cAMP increases IKsamplitude, slows its deactivation kinetics, and shifts its activation curve. At the molecular level, IKschannels are composed of KvLQT1/IsK complexes. In a variety of mammalian heterologous expression systems maintained at physiological temperature, we explored cAMP regulation of recombinant KvLQT1/IsK complexes. In these systems, KvLQT1/IsK complexes were totally insensitive to cAMP regulation. cAMP regulation was not restored by coexpression with the dominant negative isoform of KvLQT1 or with the cystic fibrosis transmembrane regulator. In contrast, coexpression of the neuronal A kinase anchoring protein (AKAP)79, a fragment of a cardiac AKAP (mAKAP), or cardiac AKAP15/18 restored cAMP regulation of KvLQT1/IsK complexes inasmuch as cAMP stimulation increased the IKsamplitude, increased its deactivation time constant, and negatively shifted its activation curve. However, in cells expressing an AKAP, the effects of cAMP stimulation on the IKsamplitude remained modest compared with those previously reported in cardiac myocytes. The effects of cAMP stimulation were fully prevented by including the Ht31 peptide (a global disruptor of protein kinase A anchoring) in the intracellular medium. We concluded that cAMP regulation of IKsrequires protein kinase A anchoring by AKAPs, which therefore participate with the channel protein complex underlying IKs.

We investigated whether high levels of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) would alter the functional properties of newly synthesized recombinant proteins. COS-7, CFPAC-1, and A549 cells were intranuclearly injected with a Simian virus 40-driven pECE-CFTR plasmid and assayed for halide permeability using the 6-methoxy- N-(3-sulfopropyl)quinolinium fluorescent probe. With increasing numbers of microinjected pECE-CFTR copies, the baseline permeability to halide dose dependently increased, and the response to adenosine 3′,5′-cyclic monophosphate (cAMP) stimulation decreased. In cells hyperexpressing CFTR, the high level of halide permeability was reduced when a cell metabolism poisoning cocktail was applied to decrease intracellular ATP and, inversely, was increased by orthovanadate. In CFPAC-1 cells investigated with the patch-clamp technique, CFTR hyperexpression led to a time-independent nonrectifying chloride current that was not sensitive to cAMP stimulation. CFPAC-1 cells hyperexpressing CFTR exhibited no outward rectifying chloride current nor inward rectifying potassium current either spontaneously or under cAMP stimulation. We conclude that hyperexpression of recombinant CFTR proteins modifies their properties inasmuch as 1) CFTR channels are permanently activated and not susceptible to cAMP regulation and 2) they lose their capacity to regulate heterologous ionic channels.