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 (5x10(-6) M) or lavendustin A (5x10(-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 G type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents, Intracellular application of myristoylated PKC substrate (5x10(-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 PIMA 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.