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 rectifyin g (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 r egulation of these currents. Under control conditions [KCl-rich pipette sol ution (1 mM free Mg2+ 10 nM free Ca2+) and KCl-rich bath solution], we foun d that IR K+ currents diminished with a t(1/2) of 7.6 min and were absent b y 30 min. Experimental manoeuvres de signed to inhibit phosphorylation incr eased the rate of current run-down. Thus, intracellular addition of 100 mu M H-7, a general kinase inhibitor, reduced the t(1/2) to 4.7 min and the cu rrents were absent by 16 min. Similarly, protein kinase A (PKA) inhibitor p eptide (50 nM) also accelerated run-down. Agents known to increase phosphor ylation, such as db-cAMP (0.5 mM) and forskolin (10 mu M), resulted in a si gnificant slowing of run-down (t(1/2) >16 min) as did intracellular additio n of the catalytic subunit of PKA (100 nM). Similarly, inhibition of dephos phorylation by either 1 mu M okadaic acid [protein phosphatase 1/2A (PP-1/2 A) inhibitor] or anti-human protein phosphatase 2C alpha (PP2C) antiserum d ecreased the rate of run-down. These results indicate that the phosphorylat ion-dependent activation state of the fetal ATII cell IR K+ channel is regu lated by a complex interplay of kinases and phosphatases involving PKA (act ivation), and PP2C and PP-1/2A (inactivation).