Abstract
Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G- proteins. pertussis toxin (PTX) (100 ng/ml) + NAP (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (P(o)) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 ± 3 picosiemens (pS) in the ± 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the P(o) of the linear 80-pS channels from 0.66 ± 0.05 to >0.9, and revealed the presence of a small (16 ± 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the P(o) of the larger conductance channel and restored PKA- sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 μM) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.
| Original language | English |
|---|---|
| Pages (from-to) | 4776-4780 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 271 |
| Issue number | 9 |
| DOIs | |
| Publication status | Published - Mar 1 1996 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes. / Ismailov, Iskander I.; Jovov, Biljana; Fuller, Catherine M.; Berdiev, Bakhram K.; Keeton, Deborah A.; Benos, Dale J.
In: Journal of Biological Chemistry, Vol. 271, No. 9, 01.03.1996, p. 4776-4780.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes
AU - Ismailov, Iskander I.
AU - Jovov, Biljana
AU - Fuller, Catherine M.
AU - Berdiev, Bakhram K.
AU - Keeton, Deborah A.
AU - Benos, Dale J.
PY - 1996/3/1
Y1 - 1996/3/1
N2 - Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G- proteins. pertussis toxin (PTX) (100 ng/ml) + NAP (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (P(o)) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 ± 3 picosiemens (pS) in the ± 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the P(o) of the linear 80-pS channels from 0.66 ± 0.05 to >0.9, and revealed the presence of a small (16 ± 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the P(o) of the larger conductance channel and restored PKA- sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 μM) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.
AB - Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G- proteins. pertussis toxin (PTX) (100 ng/ml) + NAP (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (P(o)) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 ± 3 picosiemens (pS) in the ± 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the P(o) of the linear 80-pS channels from 0.66 ± 0.05 to >0.9, and revealed the presence of a small (16 ± 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the P(o) of the larger conductance channel and restored PKA- sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 μM) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.
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U2 - 10.1074/jbc.271.9.4776
DO - 10.1074/jbc.271.9.4776
M3 - Article
VL - 271
SP - 4776
EP - 4780
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 9
ER -