Rabbit ileal villus cell brush border Na+/H+ exchange is regulated by Ca2+/calmodulin-dependent protein kinase II, a brush border membrane protein

M. E. Cohen, L. Reinlib, A. J.M. Watson, F. Gorelick, K. Rys-Sikora, M. Tse, R. P. Rood, A. J. Czernik, G. W.G. Sharp, M. Donowitz

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8990-8994
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number22
DOIs
StatePublished - 1990

Keywords

  • Na absorption
  • intestinal transport
  • phosphorylation
  • plasma membrane vesicles

ASJC Scopus subject areas

  • General

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