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

Academic Article

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.
  • Authors

    Author List

  • Berdiev BK; Shlyonsky VG; Senyk O; Keeton D; Guo Y; Matalon S; Cantiello HF; Prat AG; Ausiello DA; Ismailov II
  • Volume

  • 272
  • Issue

  • 4 41-4