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 language | English |
|---|---|
| Journal | American Journal of Physiology - Cell Physiology |
| Volume | 272 |
| Issue number | 4 41-4 |
| Publication status | Published - 1997 |
| Externally published | Yes |
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Keywords
- amiloride
- ion transport
- pertussis toxin
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology
- Physiology
- Physiology (medical)
Cite this
Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers. / Berdiev, Bakhram K.; Shlyonsky, Vadim G.; Senyk, Oksana; Keeton, Debbie; Guo, Yi; Matalon, Sadis; Cantiello, Horacio F.; Prat, Adriana G.; Ausiello, Dennis A.; Ismailov, Iskander I.; Benos, Dale J.
In: American Journal of Physiology - Cell Physiology, Vol. 272, No. 4 41-4, 1997.Research output: Contribution to journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=0030901776&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030901776&partnerID=8YFLogxK
M3 - Article
VL - 272
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
SN - 0363-6143
IS - 4 41-4
ER -