Activation of a nonspecific cation current in rat cultured retinal pigment epithelial cells: involvement of a G αi subunit protein and the mitogen‐activated protein kinase signalling pathway

1 Whole‐cell patch‐clamp recording techniques were used to investigate the G protein subtype and related signalling molecules involved in activation of a nonspecific cation (NSC) current in rat cultured retinal pigment epithelial (RPE) cells. 2 Under control conditions, in 130 m m NaCl with K + aspartate in the pipette, cytosolic dialysis with guanosine‐5′‐ O ‐(3‐triphosphate) (GTPγS, 0.1 m m ) activated a large non‐inactivating NSC current in 80% of the cells recorded from. 3 Loading RPE cells with antibodies (10 μg‐ml −1 ) against the α subunit of all PTX‐sensitive G proteins (G αi/o/t/z ) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the α subunits of the G i subclass (G αi‐3 ) completely abolished current activation. In RPE cells loaded with anti‐G αs activation of the NSC current was unaffected. 4 Investigation of the potential downstream mediators in the G αi NSC channel pathway revealed that activation of the cation conductance was unaffected by treatment of RPE cells with the selective protein kinase C inhibitor GF 109203X (3 μ m ) or the selective CaM kinase II inhibitor KN‐93 (50 μ m ). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H‐7 (100 μ m ) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 μ m ). 5 In the absence of GTPγS, the NSC current was not activated by superfusion of the cells with the cyclic GMP kinase activator dibutyryl‐cyclic GMP or with the adenylate cyclase activator forskolin. 6 These results support the involvement of a G protein of the G αi subclass in the activation of a NSC current in rat RPE cells, and suggest a potential modulatory role for MAP kinase‐dependent phosphorylation in current regulation.British Journal of Pharmacology (1998) 124 , 1115–1122; doi: 10.1038/sj.bjp.0701936