Expression and regulation of ClC-5 chloride channels: effects of antisense and oxidants.

Genetic mutations of the Cl(-) channel ClC-5 cause Dent's disease in humans. We recently cloned an amphibian ortholog of Xenopus ClC-5 (xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl(-) > I(-) anion sensitivity, and were inhibited at low pH, similar to previous results in oocytes. In oocytes, antisense xClC-5 cRNA injection had no effect on endogenous membrane currents or the heterologous expression of human ClC-5. Activators of cAMP and protein kinase C inhibitors had no significant effects on ClC-5 currents expressed in either COS-7 cells or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA) inhibitor, and hydrogen peroxide decreased the currents. We conclude that the basic properties of ClC-5 currents were independent of the host cell type used for expression. In addition, ClC-5 channels may be modulated by PKA and reactive oxygen species.

Chloride channel expression in cultured human fetal RPE cells: response to oxidative stress.

PURPOSE: The human fetal cell line RPE 28 SV4 has been useful for studies of oxidative stress and apoptosis in retinal pigmented epithelium. This cell model is now assessed in functional investigations of chloride channel activity. The study aims to determine the presence of specific chloride channels, including CFTR and ClC channels, to identify the properties of membrane chloride currents and to assess their modulation by hydrogen peroxide, cAMP, and other agents. METHODS: Channel expression was determined using RT-PCR and cDNA cloning and biochemical and immunocytochemical methods. Membrane currents were analyzed using whole-cell, patch-clamp techniques. RESULTS: RT-PCR results confirmed the presence of ClC-5 mRNA, and a full-length clone encoding ClC-3 was isolated from a cDNA library for RPE 28 SV4 cells. Specific staining for CFTR and several ClC channels was detected by immunocytochemistry. Whole-cell chloride currents (under conditions of symmetrical chloride concentrations) averaged 16.9 +/- 3.4 pA/pF (at +100 mV; n = 8), showed outward rectification, and had an anion permeability sequence of Cl(-) > I(-) > cyclamate. Currents were stimulated by cAMP cocktail (250 microM cAMP, 100 microM IBMX, and 25 microM forskolin) and were inhibited by 1 mM DIDS. The oxidative agent hydrogen peroxide (100 microM) decreased the current by 34% +/- 10% (n = 4). CONCLUSIONS. These findings suggest that RPE 28 SV4 cells possess regulated chloride channels including CFTR and members of the ClC chloride channel family. The inhibition of chloride currents by H(2)O(2) suggests that this cell line may be advantageous for studies of chloride channel modulation by oxidative stress.

Human colorectal cancers express a constitutively active cholecystokinin-B/gastrin receptor that stimulates cell growth.

Although ectopic expression of the cholecystokinin B/gastrin receptor (CCK-BR) is widely reported in human colorectal cancers, its role in mediating the proliferative effects of gastrin1-17 (G-17) on these cancers is unknown. Here we report the isolation of a novel splice variant of CCK-BR that exhibits constitutive (ligand-independent) activation of pathways regulating intracellular free Ca(2+) ([Ca(2+)](i)) and cell growth. The splice variant (designated CCK-BRi4sv for intron 4-containing splice variant) is expressed in colorectal cancers but not in normal colonic mucosa adjacent to the cancer. Balb3T3 cells expressing CCK-BRi4sv exhibited spontaneous, ligand-independent, oscillatory increases in [Ca(2+)](i), whereas cells expressing wild-type CCK-BR did not. Primary cultures of cells isolated from resected colorectal cancers also exhibited a similar pattern of spontaneous [Ca(2+)](i) oscillations. For both Balb3T3 and primary tumor cells, application of G-17 (10 and 200 nm, respectively) caused an increase in [Ca(2+)](i). Selective CCK-BR antagonists blocked the G-17-stimulated Ca(2+) responses but not the spontaneous [Ca(2+)](i) oscillations. Cells expressing CCK-BRi4sv exhibited an increased growth rate ( approximately 2.5-fold), in the absence of G-17, compared with cells expressing wild-type CCK-BR. The selective pattern of expression, constitutive activity, and trophic action associated with CCK-BRi4sv suggest that this variant may regulate colorectal cancer cell proliferation though a gastrin-independent mechanism.