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.