ACTIVATION OF A NONSPECIFIC CATION CURRENT IN RAT CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS - INVOLVEMENT OF A G(ALPHA-I) SUBUNIT PROTEIN AND THE MITOGEN-ACTIVATED PROTEIN-KINASE SIGNALING PATHWAY

1 Whole-cell patch-clamp recording techniques were used to investigate the G protein subtype and related signalling molecules involved in ac tivation of a nonspecific cation (NSC) current in rat cultured retinal pigment epithelial (RPE) cells. 2 Under control conditions, in 130 mM NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanos ine-5'-O-(3-triphosphate) (GTP gamma S, 0.1 mM) activated a large non- inactivating NSC current in 80% of the cells recorded from. 3 Loading RPE cells with antibodies (10 mu g-ml(-1)) against the alpha subunit o f all PTX-sensitive G proteins (G(alpha i/o/t/z)) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed sp ecifically against the alpha subunits of the G(i) subclass (G(alpha i- 3)) completely abolished current activation. In RPE cells loaded with anti-G(alpha s) activation of the NSC current was unaffected. 4 Invest igation of the potential downstream mediators in the G(alpha i) NSC ch annel pathway revealed that activation of the cation conductance was u naffected by treatment of RPE cells with the selective protein kinase C inhibitor GF 109203X (3 mu M) or the selective CaM kinase II inhibit or KN-93 (50 mu M). However, NSC current activation was delayed and th e current amplitude reduced in the presence of the nonselective kinase inhibitor H-7 (100 mu M) or the selective inhibitor of MAPKK (MEK) ac tivation, PD 98059 (50 mu M). 5 In the absence of GTP gamma S, the NSC current was not activated by superfusion of the cells with the cyclic GMP kinase activator dibutyryl-cyclic GMP or with the adenylate cycla se activator forskolin. 6 These results support the involvement of a G protein of the G(alpha i) subclass in the activation of a NSC current in rat RPE cells, and suggest a potential modulatory role for MAP kin ase-dependent phosphorylation in current regulation.