Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation.
We have previously shown that activity of a Cl- channel is required for acidification of clathrin-coated vesicles by the coated vesicle (H+)-ATPase (Arai, H., Pink, S. and Forgac, M. (1989) Biochemistry 28, 3075-3082). We demonstrate that activity of the coated vesicle Cl- channel is modulated by phosphorylation. Cl- conductance was measured in a reconstituted preparation of coated vesicle membrane proteins using the Cl(-)-sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium. Treatment of coated vesicle membranes with alkaline phosphatase resulted in a 25 +/- 5% decrease in Cl- channel activity. A parallel decrease in ATP-dependent acidification of coated vesicles was also observed. The decrease in Cl- conductance and ATP-dependent acidification was reversed by treatment with protein kinase A and MgATP; the alkaline phosphatase inhibitor, sodium orthovanadate, blocked the inhibition of acidification. These results indicate that Cl- conductance in coated vesicles is modulated by a protein kinase A-dependent phosphorylation and that this modulation in turn affects ATP-dependent acidification.
Mulberg
A E
AE
Department of Molecular and Cellular Physiology, Tufts University School of Medicine, Boston, Massachusetts.
Tulk
B M
BM
Forgac
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain; D002417:Cattle; D018118:Chloride Channels; D002966:Clathrin / Q000502:physiology; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000502:physiology*; D015388:Organelles / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*
D018118:Chloride Channels; D002966:Clathrin; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases