Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart

The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some other phosphatase(s). These findings are consistent with sequential phosphorylation of a protein, probably the Cl- channel itself, at two different kinds of sites. The resulting phosphoproteins can be distinguished on the basis of their different contributions to whole-cell Cl- conductance.