Protein kinase A (PKA) and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) celt 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 plus ATP increased single channel open probability (Po) from 0,42± 0.05 to 0.82 ±0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in Po of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent Ki for amiloride increased from 8.0 ±1.8 μM under control conditions to 15±3 μM after PKA-induced phosphorylation; that for ethylisopropyl amiloride increased from 1.0±0.4 μM to 2.0±0.5 μM. Neither pertussis toxin (PTX) nor GTP-γ-S 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 solubilizcd ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to the GTPγS mediated activation of the exogenous G, but not GOA, G, or G, 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 but not by G, G proteins.
|Publication status||Published - 1997|
ASJC Scopus subject areas
- Molecular Biology