Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex.
Regulation of Cl conductance by protein kinase A may play a role in control of endosomal acidification [Bae, H.-R., & Verkman, A. S. (1990) Nature, 348, 637-639]. To investigate the mechanism of kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by [gamma-32P]ATP. Endosomes had a permeability (PCl) for conductive Cl transport of 3.1 x 10(-8) cm/s at 23 degrees C which was stilbene inhibitable. PCl was increased by 90 +/- 20% by a 10-min preincubation with the catalytic subunit of kinase A (PKA, 10 units/mL) and MgATP (0.5 mM) with anion selectivity Cl greater than I greater than Br. The increase in PCl was blocked by 100 microM N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and was reversed by addition of alkaline phosphatase (AP, 40 units/mL) after incubation with PKA and MgATP; the increase in PCl was not blocked by pretreatment with AP.(ABSTRACT TRUNCATED AT 250 WORDS)
Reenstra
W W
WW
Research Institute, Children's Hospital, Oakland, California 94609.
Sabolic
I
I
Bae
H R
HR
Verkman
A S
AS
D000818:Animals; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007672:Kidney Cortex / Q000201:enzymology / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013267:Stilbenes / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D010750:Phosphoproteins; D013267:Stilbenes; D011494:Protein Kinases