Abstract
1. Changes in dihydropyridine-sensitive (L-type) Ca2+ channel kinetics were studied after prolongation of intrinsic phosphorylated time by the phosphatase inhibitor okadaic acid (OA) in cell-attached patches made from single isolated rabbit ventricular myocytes, using barium as the charge carrier. 2. At low concentrations (0.001-1 microM), OA decreased the number of sweeps without openings, while open duration was not changed. However, when cells were pretreated by a membrane-permeant cyclic AMP, 0.1 microM OA induced long-lasting channel openings as well. 3. At high concentrations (10-750 microM), OA additionally induced long-lasting openings, resulting in open time distributions that were best fitted by two exponentials. 4. The durations of an available state (TS) and an unavailable state (TF) were estimated by the numbers of non-blank sweeps per run and blank sweeps per run by applying repetitive 45 ms steps at 2 Hz to 0 mV from holding potentials of -80 mV. TS was well fitted by an exponential curve, of which the time constant was increased from 0.67 to 1.60 sweeps by 0.1 microM OA, while TF was 0.347 sweeps and remained unchanged. 5. OA activated brief openings and long-lasting, wide openings in a concentration-dependent manner. Namely, we find different dose-response relationships for the two kinetic effects of increased opening probability (mode 1) and prolongation of opening (mode 2). This behaviour suggests that there are at least two modulatory phosphorylation sites that are dephosphorylated by different phosphatases.