CAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

Abstract

The role of adenosine 3',5'-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl- conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100 nM catalytic subunit of PK-A purified from human placenta and 1 mM of the phosphatase resistant adenosine 5'-thiotriphosphate (ATP-gamma-S) and resealed. Cl- conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23 degrees C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([14C]-sucrose) and included in each Cl- flux calculation. In 19 independent experiments, the mean Cl- conductance in placental membranes in the absence of phosphorylation was 3.67 +/- 3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97 +/- 1.75 nmol.sec-1. (mg protein)-1 (mean +/- SD). PK-A dependent phosphorylation reduced the Cl- conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl- channels, confirmed that the PK-A dependent phosphorylation increased in Cl- conductance in 11/13 experiments, from 1.01 +/- 0.61 to 1.85 +/- 0.99 nmol.sec-1.(mg protein)-1 (mean +/- SD). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl- channel(s) of the apical membranes of human placenta.