Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Abstract

In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl- absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphorprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45 +/- 13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on the Vmax and not on the Km for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120 nM. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. We conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.

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

“…Brush border membranes, consisting predominantly of closed vesicles, were made as described (7,8) starting with Proc. Natl.…”

“…Constituents necessary for regulation of Ca2+/CaM-dependent protein kinase activity were incorporated into the brush border vesicles by freezing and thawing the vesicles as described (7,8 (Fig. 1).…”

“…These studies were largely based on use of Ca2+/CaM antagonists, which have multiple actiofls. Furthermore, enclosure of Ca2', CaM, and ATP into right-side-out brush border vesicles from ileal villus cells blocks Na+/H+ exchange (7,8). In addition, an endogenous CaM kinase is present in ileal villus cell brush border membranes, which is active at basal levels of [Ca2W]i (9).…”

Calmodulin Kinase II Constitutively Binds, Phosphorylates, and Inhibits Brush Border Na+/H+ Exchanger 3 (NHE3) by a NHERF2 Protein-dependent Process

“…We previously showed that CaMKII is present in small intestinal BB where it inhibits neutral NaCl absorption under basal conditions, and studied in isolated ileal villus cell BB, it inhibits NHE3 activity by a process consistent with phosphorylation, because it was Mg 2ϩ -, ATP-, Ca 2ϩ -, and calmodulindependent (21,34). The similarity of the CaMKII effects in intact ileal Na ϩ -absorptive cells, isolated absorptive cell BB, PS120 fibroblasts, and Caco-2-BBe cells, as shown here, supports that this inhibition of basal NHE3 activity by CaMKII is intrinsic to NHE3 rather than being tissue-or cell-specific.…”

“…We previously demonstrated that CaMKII inhibits ileal neutral NaCl absorption and its constituent BB Na ϩ /H ϩ exchanger under basal conditions (21,34). In examining the mechanism for this effect, we reported that pretreatment of PS120 cells stably expressing NHE3 with the CaMKII inhibitor KN-62 increased Na ϩ /H ϩ exchange activity (22).…”