ACTIVATION OF A CL--CONDUCTANCE BY PROTEIN KINASE-DEPENDENT PHOSPHORYLATION IN CULTURED RAT RETINAL-PIGMENT EPITHELIAL-CELLS

While chloride conductances are involved in signals of the electroreti nogram generated by the retinal pigment epithelium (RPE), patch-clamp experiments of freshly isolated or cultured RPE cells have shown that potassium conductances predominate. The purpose of this study was to i nvestigate mechanisms which activate Cl--conductances in RPE cells. Me mbrane currents of cultured rat RPE cells were measured using the whol e-cell configuration of the patch-clamp technique under extra-and intr acellular K+-free conditions. The bath solution was hyperosmolal to th e pipette solution to prevent hypoosmotic swelling. Exchange of the ph ysiological intracellular fluid by a pipette solution with physiologic al levels of ATP (2 mM) induced a continuous increase of membrane cond uctance. Conductance was blocked by DIDS (1 mM), and showed a reversal potential close to the Nernst potential for Cl-. When the experiments were carried out under conditions in which all cations, and not only potassium, were replaced by NMDG, the same responses could be observed . Current activation was independent of extracellular calcium. Chlorid e currents were also induced when ATP gamma S or AMP-PNP were used ins tead of ATP. In the presence of AMP-PNP currents were 10 times smaller than in the presence of ATP or ATP gamma S. In cells preincubated wit h staurosporine or chelerythrine no currents were induced. Establishin g the whole-cell configuration with ATP and with myristoylated PKC sub strate in addition, no voltage-dependent currents were activated. We c onclude that ATP hydrolysis leads to activation of chloride currents v ia PKC in the whole-cell configuration. The perforated patch configura tion, with the intracellular compartment intact, no currents were indu ced under otherwise identical experimental conditions. Inhibition of p hosphatase by calyculin (10 nM) in the perforated-patch configuration did not change membrane conductance. In the intact cell, chloride cond uctance is possibly inhibited by a cytosolic factor which is washed ou t when the whole-cell configuration is established. (C) 1998 Academic Press Limited.