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

Bakhram K. Berdiev, Vadim G. Shlyonsky, Oksana Senyk, Debbie Keeton, Yi Guo, Sadis Matalon, Horacio F. Cantiello, Adriana G. Prat, Dennis A. Ausiello, Iskander I. Ismailov, Dale J. Benos

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

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 pM under control conditions to 15 ± 3 pM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 ± 0.4 to 2.0 ± 0.5 μM. 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 Gα(i-2), but not G(oA), Gα(i-1), or G{ai-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 Gα(i-2), but not Gα(i-1) or Gα(i-3), protein.

Original languageEnglish
Pages (from-to)C1262-C1270
JournalAmerican Journal of Physiology - Cell Physiology
Volume272
Issue number4 41-4
DOIs
Publication statusPublished - 1997

Keywords

  • amiloride
  • ion transport
  • pertussis toxin

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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