Protein kinase a phosphorylation and g-protein regulation of type
ii pneumocyte na+ channels in lipid bilayers.
Berdiev, Bakhram K., Vadim Gh. Shlyonsky, Oksana Senyk, Debbie Keeton,
Yi Guo+, Sadis Matalon, Horacio F. Cantiello, Adriana G. Prat, Dennis
A. Ausiello, Iskander I. Ismailov, and Dale J. Benos.
Departments of Physiology and Biophysics, Pulmonary Medicine, and
Anesthesiology, The University of Alabama at Birmingham, Birmingham,
Alabama 35294 Renal Unit, Massachusetts General Hospital,
Charlestown, Massachusetts 02129
APStracts 3:0402C, 1996.
Protein kinase A 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 protein kinase A (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 [mu]M under
control conditions to 15 +/- 3 [mu]M after PKA-induced
phosphorylation; that for ethylisopropyl amiloride increased from 1.0
+/- 0.4 [mu]M to 2.0 +/- 0.5 [mu]M. Neither pertussis toxin nor GTP
-[delta]-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 solubilized ATII cell protein fraction, but not in the
immunopurified fraction. Moreover, the immunopurified channel was
downregulated in response to the GTP[delta]S mediated activation of
the exogenous Gi[alpha]2, but not GOA, Gi[alpha]1, or Gi[alpha]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 Gi[alpha]2
but not by Gi[alpha]1, Gi[alpha]3 proteins.
Received 11 January 1996; accepted in final form 14 November
1996.
APS Manuscript Number C16-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996