Rabbit ileal villus cell brush border Na+/H+ exchange is regulated by Ca2+/calmodulin-dependent protein kinase II, a brush border membrane protein.
Ileal brush border membranes contain an endogenous Ca2+/calmodulin (CaM)-dependent protein kinase activity that modulates the activity of the apical membrane Na+/H+ exchanger. To further characterize this kinase, synapsin I, a substrate for Ca2+/CaM-dependent protein kinases, was added to preparations of ileal brush border membranes. In the presence of Ca2+/CaM, synapsin I was phosphorylated. Phosphopeptide mapping demonstrated that the addition of Ca2+/CaM to brush border membranes stimulated the phosphorylation of sites in synapsin I specific for Ca2+/CaM-dependent protein kinase II. Immunoblots containing brush border and microvillus membrane proteins were probed with an antibody that recognizes the 50-kDa subunit of rat brain Ca2+/CaM-dependent protein kinase II. This antibody labeled major and minor species of 50 and 53 kDa, respectively, with more labeling of the brush border than the microvillus membranes. Right-side-out ileal villus cell brush border vesicles were prepared containing CaM, ATP, and 350 nM free Ca2+. Na+/H+ exchange was inhibited by the presence of Ca2+/CaM/ATP within the vesicles. A 21-amino acid peptide inhibitor of CaM kinase II was enclosed within some vesicle preparations by freeze-thaw. The effect on Na+/H+ exchange of Ca2+/CaM/ATP was partially reversed by the inhibitor peptide. These studies demonstrate the presence of Ca2+/CaM-dependent protein kinase II in rabbit ileal villus cell brush border membranes. Based on the effect of a specific inhibitor peptide of Ca2+/CaM kinase II, it is concluded that this kinase inhibits brush border Na+/H+ exchange, which participates in the regulation of ileal Na+ absorption.
Cohen
M E
ME
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Reinlib
L
L
Watson
A J
AJ
Gorelick
F
F
Rys-Sikora
K
K
Tse
M
M
Rood
R P
RP
Czernik
A J
AJ
Sharp
G W
GW
Donowitz
M
M
D000818:Animals; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002352:Carrier Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000378:metabolism; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007408:Intestinal Absorption; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D016704:Synapsins
D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D017923:Sodium-Hydrogen Exchangers; D016704:Synapsins; D012964:Sodium; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium