CAMP-mediated inhibition of the renal brush border membrane Na+-H+ exchanger requires a dissociable phosphoprotein cofactor.
Prior studies have suggested that protein kinase A (PKA)-mediated inhibition of the rabbit renal brush border membrane (BBM) Na(+)-H+ exchanger involves a regulatory protein that is distinct from the transporter. This putative regulatory protein was purified by column chromatography and SDS-PAGE, and a partial primary amino acid sequence was determined. An affinity-purified polyclonal antibody to a synthetic peptide representing a sequence of the protein recognized a polypeptide of 55 kD in BBM but not in basolateral membrane. The antibody immunoprecipitated a PKA substrate of a similar molecular mass from detergent-solubilized BBM proteins. When assayed after reconstitution, PKA in the presence of ATP and Mg2+ did not inhibit Na(+)-H+ exchange transport in a fraction of solubilized BBM proteins eluting from an anion exchange column between 0.2 and 0.4 M NaCl (fraction B). Coreconstitution of fraction B with the immunoprecipitated 55-kD protein restored the inhibitory effect of PKA (change = 42%, P < 0.05). By contrast, Na(+)-H+ exchange transport in total solubilized BBM proteins was inhibited 25% (P < 0.05) by PKA, ATP, and Mg2+. This effect was abolished by immunodepletion of the cAMP regulatory protein (change = +5%, P = NS). These findings provide evidence that the regulation of renal BBM Na(+)-H+ exchange transport by PKA is affected by repletion and depletion of a specific protein. This suggests that PKA-mediated inhibition of the renal BBM Na(+)-H+ exchanger requires participation of a regulatory protein that is distinct from the transporter itself.
Weinman
E J
EJ
Department of Medicine, University of California, Los Angeles School of Medicine.
Steplock
D
D
Shenolikar
S
S
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry
D008081:Liposomes; D008565:Membrane Proteins; D010750:Phosphoproteins; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers; C020485:proteoliposomes; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015293:Transducin