TY - JOUR
T1 - Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers
AU - Berdiev, Bakhram K.
AU - Shlyonsky, Vadim G.
AU - Senyk, Oksana
AU - Keeton, Debbie
AU - Guo, Yi
AU - Matalon, Sadis
AU - Cantiello, Horacio F.
AU - Prat, Adriana G.
AU - Ausiello, Dennis A.
AU - Ismailov, Iskander I.
AU - Benos, Dale J.
PY - 1997
Y1 - 1997
N2 - 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.
AB - 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.
KW - amiloride
KW - ion transport
KW - pertussis toxin
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U2 - 10.1152/ajpcell.1997.272.4.c1262
DO - 10.1152/ajpcell.1997.272.4.c1262
M3 - Article
C2 - 9142851
AN - SCOPUS:0030901776
VL - 272
SP - C1262-C1270
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
SN - 0363-6143
IS - 4 41-4
ER -