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 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 ATII cells. Addition of the catalytic 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 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 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(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. 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 G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.
Berdiev
B K
BK
Department of Physiology and Biophysics, University of Alabama at Birmingham, 35294, USA.
Shlyonsky
V G
VG
Senyk
O
O
Keeton
D
D
Guo
Y
Y
Matalon
S
S
Cantiello
H F
HF
Prat
A G
AG
Ausiello
D A
DA
Ismailov
I I
II
Benos
D J
DJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D008051:Lipid Bilayers / Q000378:metabolism; D008297:Male; D037342:Pertussis Toxin; D010766:Phosphorylation; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008051:Lipid Bilayers; D015222:Sodium Channels; D010566:Virulence Factors, Bordetella; D000584:Amiloride; D037342:Pertussis Toxin; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins