Inwardly rectifying K+ currents in fetal alveolar type II cells: regulation by protein kinase A and protein phosphatases

Fetal guinea-pig lung alveolar type II (ATII) cells have inwardly rectifying (IR) K+ currents that display Mg2+- and G-protein-dependent run-down. We have used the whole-cell patch-clamp technique to investigate further the regulation of these currents. Under control conditions [KCl-rich pipette solution (1 mM free Mg2+, 10 nM free Ca2+) and KCl-rich bath solution], we found that IR K+ currents diminished with a t1/2 of 7.6 min and were absent by 30 min. Experimental manoeuvres designed to inhibit phosphorylation increased the rate of current run-down. Thus, intracellular addition of 100 microM H-7, a general kinase inhibitor, reduced the t1/2 to 4.7 min and the currents were absent by 16 min. Similarly, protein kinase A (PKA) inhibitor peptide (50 nM) also accelerated run-down. Agents known to increase phosphorylation, such as db-cAMP (0.5 mM) and forskolin (10 microM), resulted in a significant slowing of run-down (t1/2>16 min) as did intracellular addition of the catalytic subunit of PKA (100 nM). Similarly, inhibition of dephosphorylation by either 1 microM okadaic acid [protein phosphatase 1/2A (PP-1/2A) inhibitor] or anti-human protein phosphatase 2Calpha (PP2C) antiserum decreased the rate of run-down. These results indicate that the phosphorylation-dependent activation state of the fetal ATII cell IR K+ channel is regulated by a complex interplay of kinases and phosphatases involving PKA (activation), and PP2C and PP-1/2A (inactivation).