1. Application of the phosphatase inhibitors okadaic acid (OA) and microcystin (MC) to frog cardiomyocytes caused large increases in L-type calcium current (I(Ca)) in the absence of beta-adrenergic agonists. The increase occurred without effects on the peak current-voltage relation or voltage-dependent inactivation. OA and MC caused a decrease in amplitude of delayed rectifier current (I(K)), which is opposite to the increase produced by cAMP-dependent phosphorylation. The decrease occurred without effects on voltage-dependent activation or reversal potential. 2. Analysis of the dose-response relations for OA and MC on ventricular cell I(Ca) were best fitted with a single-site relationship with 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 (calcineurin) was without appreciable effect. 3. Reducing intracellular ATP levels was without effect on basal I(Ca) suggesting that calcium channels may not need to be phosphorylated to open. ATP depletion was able to block completely the I(Ca) increase induced by OA or MC. This demonstrates that the effects of OA and MC on I(Ca) are mediated by a phosphorylation reaction. In contrast, ATP depletion totally abolished I(K), suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier channel. 4. Internal perfusion of a peptide inhibitor (PKI(5-22)) of protein kinase A (PK-A) was without effect on basal current levels of I(Ca) or I(K), suggesting that this kinase is not phosphorylating these channels under basal conditions. Furthermore, although PKI is capable of completely blocking the response of I(Ca) to isoprenaline or forskolin, PKI does not affect the increase in I(Ca) induced by MC or OA. Inhibition of adenylate cyclase with acetylcholine or inhibition of PK-A with adenosine cyclic 3',5'-(Rp)--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 complete phosphorylation of PK-A sites. 6. To attempt to identify the protein kinase activity responsible for OA effects on I(Ca) and I(K), several types of protein kinase inhibitors were internally perfused. Inhibitors, including those for PK-A, protein kinase C, calcium-calmodulin-dependent protein kinase II, tyrosine kinase, and cGMP-dependent kinases were all without effect on OA responses or on basal current levels. 7. These results suggest that an unidentified, basally active protein kinase(s) is capable of phosphorylating and stimulating I(Ca). These activities are apparently nullified by a basally active protein phosphatase. Furthermore, the delayed rectifier channel in frog atrial cells apparently has multiple phosphorylation sites, one that must be phosphorylated for basal channel function, which may be the same as the one stimulated by PK-A, and a second one which inhibits channel activity and is revealed by inhibition of protein phosphatase activity.
