Abstract
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.
Under control conditions, in 130 mM NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanosine-5′-O-(3-triphosphate) (GTPγS, 0.1 mM) activated a large non-inactivating NSC current in 80% of the cells recorded from.
Loading RPE cells with antibodies (10 μg-ml−1) against the α subunit of all PTX-sensitive G proteins (Gαi/o/t/z) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the α subunits of the Gi subclass (Gαi-3) completely abolished current activation. In RPE cells loaded with anti-Gαs activation of the NSC current was unaffected.
Investigation of the potential downstream mediators in the Gαi 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 μM) or the selective CaM kinase II inhibitor KN-93 (50 μM). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H-7 (100 μM) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 μM).
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.
These results support the involvement of a G protein of the Gαi 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.
Keywords: Retinal pigment epithelium, nonspecific cation current, GTPγS, G protein, MAP kinase
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