Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers

BAKHRAM K. BERDIEV, VADIM G. SHLYONSKY, OK&WA SENYK, DEBBIE KEETON, YI GU0, SADIS MATALON, HORACIO F. CANTIELLO, ADRIANA G. PRAT, DENNIS A. AUSIELL0, ISKANDER I. ISMAILOV AND DALE J. BENOS

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY

Año: 1997 p. 1262 - 1270

0363-6143

Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers. Am. J. Physiol. 272 (CeZZ Physiol. 41): C1262-Cl270, 1997.-Protein kinase A (PKA)-and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated AT11 cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 t 0.05 to 0.82 t, 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 or 1.8 uM under control conditions to 15 t 3 uM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 t 0.4 to 2.0 t 0.5 uM. Neither pertussis toxin (PTX) nor guanosine 5’.0-(3-thiotriphosphate) affected AT11 Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride- inhibitable 22Na+ uptake in freshly isolated AT11 cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized AT11 cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5’-0-(3-thiotriphosphate)- mediated activation of the exogenous Gtii-2, but not Gd, GoLi-1, or hi-39 protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by Gai-2, but not Goi- or Goiprotein.