Regulation of the rabbit ileal brush-border Na⁺/H⁺ exchanger by an ATP-requiring Ca⁺⁺/calmodulin-mediated process

Brush-border vesicles purified from rabbit ileal villus cells were used to evaluate how Ca⁺⁺/calmodulin (CaM) regulates the neutral linked NaCl absorptive process, part of which is a Na⁺/H⁺ exchanger. After freezing and thawing to allow incorporation of macromolecules into the vesicles, the effect of Ca⁺⁺/CaM on brush-border Na⁺ uptake with an acid inside pH gradient, and on Na⁺/H⁺ exchange was determined. Freezing and thawing vesicles with 0.85 μM free Ca⁺⁺ plus 5 μM exogenous CaM failed to alter Na⁺/H⁺ exchange as did the addition of exogenous ATP plus an ATP regenerating system, which was sufficient to elevate intravesicular ATP to 47 μM from a basal level of 0.4 μM. However, the combination of Ca⁺⁺/CaM plus ATP inhibited Na⁺ uptake in the presence of an acid inside pH gradient and inhibited Na⁺/H⁺ exchange, while Na⁺ uptake in the absence of a pH gradient was not altered. This effect required a hydrolyzable form of ATP, and did not occur when the nonhydrolyzable ATP analogue, AMP-PNP, replaced ATP. Under the identical intravesicular conditions used for the transport studies, Ca⁺⁺ (0.85 μM) plus exogenous CaM (5 μM), in the presence of magnesium plus ATP, increased phosphorylation of five brush-border peptides. These data are consistent with Ca⁺⁺/CaM acting via phosphorylation to regulate the ileal brush-border Na⁺/H⁺ exchanger.