Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium

Abstract

little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H2O2. The inhibitory effects of H2O2 were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.immunocytochemistry; patch clamp; glutathione; glutathione S-transferase RETINAL PIGMENT EPITHELIUM (RPE) comprises part of the blood-retina barrier and functions in several processes that are vital for the preservation of sight. Among the crucial roles of this epithelium is the regulation of the volume and electrolyte composition of the subretinal space. This function is achieved largely by regulated transepithelial transport of chloride from the subretinal space to the choroid with the obligatory movement of water. Disruption of RPE chloride transport can result in the accumulation of fluid in the subretinal space and subsequent retinal detachment (3, 26).Chloride transport across the RPE is mediated in part by chloride channels that are stimulated by calcium and cAMP (Ref. 28; cf. Refs. 13,14). In recent whole cell patch-clamp recordings in SV40-transformed cultured human fetal RPE (hfRPE) cells, we (39) identified an outwardly rectifying chloride current that was stimulated by cAMP but was inhibited by the chloride channel blocker DIDS, acidic bathing solutions, or low concentrations of the oxidative agent H 2 O 2 . The molecular identity of the chloride channel(s) responsible for this current was not determined. However, these cells expressed several candidate chloride channels including cystic fibrosis transmembrane conductance receptor (CFTR) and members of the ClC chloride channel family Ref. 39). At present, little is known about the expression of ClC chloride channels in the intact human RPE.Recent evidence suggests that the ClC family of voltage-gated chloride channels may be crucial for retinal function. At least three members of this family are associated with retinal degenerations in mice. Specifically, transgenic mice that were deficient for ClC-3 (34), ClC-2 (6), or ClC-7 (17) were found to develop retinal degenerations and blindness within weeks after birth. The basis of these degenerations is not presently understood.The finding of ClC channel expression in human fetal cells and the consequences of ...

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

“…These currents exhibited very steep outward recti- fication, reversed near the Cl Ϫ equilibrium potential, were inhibited by removal of extracellular Cl Ϫ , and displayed slow time-dependent activation. These basic properties were strikingly similar to those of published ClC-4 (16) and ClC-5 (16,53) currents. Over the physiological pH range, changes in current amplitude and reversal potential in response to altered extracellular Cl Ϫ and H ϩ concentrations were consistent with ClC-3 acting as a proton-anion exchanger, similar to ClC-4 and -5 (39,44).…”

S-CMC-Lys Protective Effects on Human Respiratory Cells During Oxidative Stress

“…Inhibitory or stimulatory effects of ROS on anion channels have been evidenced [17,[24][25][26][27][28][29][30][31]. Here we shown tor in affecting cell vulnerability or tolerance to oxidative insults, and it has been implicated in immune modulation and inflammatory responses [36].…”

Mitochondrial Fatty Acid β-Oxidation in the Human Eye and Brain: Implications for the Retinopathy of Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency

“…Of these cell layers, the RPE, nonpigmented ciliary epithelium, and choroid plexus contain high concentrations of mitochondria, consistent with a central role for oxidative phosphorylation in metabolism of these cells. There is also increasing evidence that the RPE cells are rich in ion channels (21)(22)(23)(24). Coma and brain edema have been detected in patients with ␤-oxidation defects, even during normoglycemia (25).…”