Chloride currents in acutely isolated Xenopus retinal pigment epithelial cells

Abstract

The retinal pigment epithelium (RPE) regulates the ionic composition of the fluid surrounding the photoreceptors by transport mechanisms that utilize Cl⁻ channels. Cl⁻ currents in RPE cells, however, remain incompletely characterized. The purpose of this study was to identify the Cl⁻ currents in acutely isolated Xenopus RPE cells using whole-cell patch clamp. We describe three different Cl⁻ currents. (1) An inwardly rectifying Cl⁻ current, I_Cl,ir, activates slowly with hyperpolarization (τ_act=μ1 s at −80 mV, V_1/2=−94 ± 3 mV), is blocked by Zn²⁺ (IC₅₀=185 μm), is stimulated by acid (I_Cl,ir is 5 times larger at pH 6 than pH 8), and is blocked by DIDS in a voltage-dependent manner. I_Cl,ir closely resembles cloned ClC-2currents. (2) An outwardly rectifying Cl⁻ current, I_Cl,Ca, is stimulated by elevated cytosolic free [Ca²⁺]. With 1 μm free [Ca²⁺]_i in the patch pipette, I_Cl,Ca activates slowly with depolarization (τ_act=325 ms at 100 mV) and deactivates upon hyperpolarization. I_Cl,Ca is not blocked by 1 mm Zn²⁺ or 10 μm Gd³⁺ but is blocked by DIDS. High extracellular [Ca²⁺] (10 mm) also activates I_Cl,Ca. (3) A non-rectifying current is activated by elevation of cytoplasmic cAMP with forskolin and IBMX. In addition to these three Cl⁻ currents, Xenopus RPE cells exhibit a non-selective background current (I_bkg) which has a linear I-V relationship and is voltage insensitive. This current is blocked by Zn²⁺ (IC₅₀ of 5.3 μm) or 10 μm Gd³⁺. This description provides new insights into the physiology of Cl⁻ channels involved in salt and fluid transport by the retinal pigment epithelium.