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 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 mM NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanosine-5'-O-(3-triphosphate) (GTPgammaS, 0.1 mM) activated a large non-inactivating NSC current in 80% of the cells recorded from. 3. Loading RPE cells with antibodies (10 microg-ml(-1)) against the alpha subunit of all PTX-sensitive G proteins (G(alpha i/o/t/z)) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the alpha subunits of the Gi 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. Investigation of the potential downstream mediators in the G(alpha i) 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 microM) or the selective CaM kinase II inhibitor KN-93 (50 microM). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H-7 (100 microM) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 microM). 5. In the absence of GTPgammaS, 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(alpha i) 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.
Ryan
J S
JS
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
Kelly
M E
ME
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002412:Cations; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans
D002412:Cations; D004791:Enzyme Inhibitors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D006152:Cyclic GMP