PROTEIN-KINASE-A PHOSPHORYLATION AND G-PROTEIN REGULATION OF TYPE-II PNEUMOCYTE NA+ CHANNELS IN LIPID BILAYERS

Protein kinase A (PKA)- and G protein-mediated regulation of immunopur ified adult rabbit alveolar epithelial type II (ATII) cell proteins th at exhibit amiloride-sensitive Na+ channel activity was studied in pla nar lipid bilayers and freshly isolated ATII cells. Addition of the ca talytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probabili ty of the channels was mainly due to a decrease in the time spent by t he channel in its closed state. The apparent inhibition constant for a miloride increased from 8.0 +/- 1.8 mu M under control conditions to 1 5 +/- 3 mu M after PKA-induced phosphorylation; that for ethylisopropy lamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 mu M. Neither per tussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected AT II Na+ channel activity in bilayers. Moreover, PTX failed to affect am iloride-inhibitable Na-22(+) uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a spe cifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreove r, the immunopurified channel was downregulated in response to guanosi ne 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G al pha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. Th ese results suggest that amiloride-sensitive Na+ channels in adult alv eolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha(i-2), but not G alpha(i-1) or G alpha(i-3), protein.