OPPOSITE EFFECTS OF PHOSPHATASE INHIBITORS ON L-TYPE CALCIUM AND DELAYED RECTIFIER CURRENTS IN FROG CARDIAC MYOCYTES

1. Application of the phosphatase inhibitors okadaic acid (OA) and mic rocystin (MC) to frog cardiomyocytes caused large increases in L-type calcium current (I(Ca)) in the absence of beta-adrenergic agonists. Th e increase occurred without effects on the peak current-voltage relati on or voltage-dependent inactivation. OA and MC caused a decrease in a mplitude of delayed rectifier current (I(K)), which is opposite to the increase produced by cAMP-dependent phosphorylation. The decrease occ urred without effects on voltage-dependent activation or reversal pote ntial. 2. Analysis of the dose-response relations for OA and MC on ven tricular cell I(Ca) were best fitted with a single-site relationship w ith a K1/2 of 1.58 mum and 0.81 muM, respectively. These data suggest the predominant form of phosphatase active on I(Ca) in this cell type is produced by protein phosphatase 1. Inhibition of phosphatase 2B (ca lcineurin) was without appreciable effect. 3. Reducing intracellular A TP levels was without effect on basal I(Ca) suggesting that calcium ch annels may not need to be phosphorylated to open. ATP depletion was ab le to block completely the I(Ca) increase induced by OA or MC. This de monstrates that the effects of OA and MC on I(Ca) are mediated by a ph osphorylation reaction. In contrast, ATP depletion totally abolished I (K), suggesting either a requirement for ATP or phosphorylation for ba sal function of the delayed rectifier channel. 4. Internal perfusion o f a peptide inhibitor (PKI(5-22)) of protein kinase A (PK-A) was witho ut effect on basal current levels of I(Ca) or I(K), suggesting that th is kinase is not phosphorylating these channels under basal conditions . Furthermore, although PKI is capable of completely blocking the resp onse of I(Ca) to isoprenaline or forskolin, PKI does not affect the in crease in I(Ca) induced by MC or OA. Inhibition of adenylate cyclase w ith acetylcholine or inhibition of PK-A with adenosine cyclic 3',5'-(R p)--phosphothioate (R(p)-cAMPS) also had no effect on the response to OA or MC. 5. Application of beta-adrenergic agonist, forskolin or cAMP all produced additional increases in the presence of saturating doses of MC or OA. This supports the hypothesis that PK-A is not mediating the OA response and that phosphatase inhibition does not result in com plete phosphorylation of PK-A sites. 6. To attempt to identify the pro tein kinase activity responsible for OA effects on I(Ca) and I(K), sev eral types of protein kinase inhibitors were internally perfused. Inhi bitors, including those for PK-A, protein kinase C, calcium-calmodulin -dependent protein kinase II, tyrosine kinase, and cGMP-dependent kina ses were all without effect on OA responses or on basal current levels . 7. These results suggest that an unidentified, basally active protei n kinase(s) is capable of phosphorylating and stimulating I(Ca). These activities are apparently nullified by a basally active protein phosp hatase. Furthermore, the delayed rectifier channel in frog atrial cell s apparently has multiple phosphorylation sites, one that must be phos phorylated for basal channel function, which may be the same as the on e stimulated by PK-A, and a second one which inhibits channel activity and is revealed by inhibition of protein phosphatase activity.