Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells.
The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.
Strauss
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O
Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin der Freien Universität Berlin, Germany.
Mergler
S
S
Wiederholt
M
M
D000818:Animals; D015220:Calcium Channels / Q000502:physiology*; D020746:Calcium Channels, L-Type; D002478:Cells, Cultured; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate