Mouse retinal pigment epithelial cells exhibit a thiocyanate-selective conductance

Abstract

The basolateral membrane anion conductance of the retinal pigment epithelium (RPE) is a key component of the transepithelial Cl transport pathway. Although multiple Cl channels have been found to be expressed in the RPE, the components of the resting Cl conductance have not been identified. In this study, we used the patch-clamp method to characterize the ion selectivity of the anion conductance in isolated mouse RPE cells and in excised patches of RPE basolateral and apical membranes. Relative permeabilities ( P/ P) calculated from reversal potentials measured in intact cells under bi-ionic conditions were as follows: SCN >> ClO > [Formula: see text] > I > Br > Cl >> gluconate. Relative conductances ( G/ G) followed a similar trend of SCN >> ClO > [Formula: see text] > I > Br ≈Cl >> gluconate. Whole cell currents were highly time-dependent in 10 mM external SCN, reflecting collapse of the electrochemical potential gradient due to SCN accumulation or depletion intracellularly. When the membrane potential was held at -120 mV to minimize SCN accumulation in cells exposed to 10 mM SCN, the instantaneous current reversed at -90 mV, revealing that P/ P is approximately 500. Macroscopic current recordings from outside-out patches demonstrated that both the basolateral and apical membranes exhibit SCN conductances, with the basolateral membrane having a larger SCN current density and higher relative permeability for SCN. Our results suggest that the RPE basolateral and apical membranes contain previously unappreciated anion channels or electrogenic transporters that may mediate the transmembrane fluxes of SCN and Cl.

Electrophysiological impact of thiocyanate on isolated mouse retinal pigment epithelial cells

“…Dependence of currents in 500 M external SCN Ϫ on holding potential. Previously, we showed that in the presence of 10 mM or 140 mM external SCN Ϫ , the amplitude, kinetics, and reversal potential of whole cell currents in mouse RPE cells depended on the holding potential (7). These effects were also observed when the external SCN Ϫ concentration was reduced to 500 M. Figure 2 shows representative families of currents evoked by a series of voltage steps from holding potentials of 0 mV (Fig.…”

“…Concentration dependence of SCN Ϫ currents. We recently showed that, when exposed to 10 mM external SCN Ϫ , voltageclamped mouse RPE cells exhibited prominent transient currents whose large negative reversal potential indicates that the anion conductance of the RPE cell membranes has a remarkably high relative permeability for SCN Ϫ (P SCN /P Cl ) of Ն500 (7). To determine whether the RPE cell is sensitive to more physiologically relevant concentrations of SCN Ϫ , we recorded whole cell currents under voltage clamp conditions in C57BL/6J mouse RPE cells exposed to extracellular SCN Ϫ in the range of 50 to 500 M. Figure 1 shows families of whole cell currents evoked by voltage steps from a holding potential (HP) of 0 mV in an RPE cell exposed to 0 M SCN Ϫ (Fig.…”

“…Enucleated eyes were hemisected posteriorly to the ora serrata, and the anterior segment and lens were removed. RPE cells were then isolated enzymatically using papain as described previously (7), stored in L-15 medium containing 0.5 mM taurine and 1 mM reduced glutathione at 4°C, and used within 8 h. All solutions used for the isolation and storage of RPE cells were bubbled with 90% N 2/10% O2. This measure improved cell quality significantly.…”

“…4D, top right). The two components to the time-dependent decay in currents may reflect the accumulation or depletion of SCN Ϫ taking place in two cellular compartments: a relatively small extracellular compartment corresponding to basal membrane infoldings wherein flux-associated SCN Ϫ concentration changes occur rapidly and a larger compartment consisting of the cytoplasm where SCN Ϫ concentration changes develop more slowly (7).…”

“…The retinal pigment epithelium (RPE) is a transporting epithelium that supports optimal visual function by tightly regulating the volume and ionic composition of the extracellular fluid surrounding the inner and outer segments of rod and cone photoreceptors. We recently discovered that mouse RPE cells have a large conductance and permeability for thiocyanate (SCN Ϫ ) (7). This conductance, which lies predominantly in the basolateral membrane, exhibits an anion selectivity profile as well as other properties that do not match those of any of the Cl Ϫ channels reported to be expressed in the RPE, namely ANO1 (8), ANO2 (15), BEST1 (17), CFTR (4), and ClC-2 (5).…”