Characterization of calcium transport by basolateral membrane vesicles of human small intestine

The present studies investigated the mechanism of Ca²⁺ transport across basolateral membrane vesicles (BLMVs) prepared from human small intestine. Ca²⁺ uptake represented transport into the intravesicular space as evident by osmolality study and by the demonstration of Ca²⁺ efflux from the intravesicular space by Ca²⁺ ionophore A23187. Ca²⁺ uptake was stimulated by Mg²⁺-ATP. Kinetic parameters for ATP-dependent Ca²⁺ uptake revealed a Michaelis constant (K(m)) of 0.02 ± 0.01 μM and a maximum rate of uptake (V(max)) of 1.00 ± 0.03 nmol·mg protein^-1·min^-1. Ca²⁺ uptake in the absence of Mg²⁺ was inhibited by 75%. The K(m) of ATP concentration required for half-maximal Ca²⁺ uptake was 0.50 ± 0.1 mM. Calmodulin (10 μg/ml) increased V(max) to 1.62 ± 0.02 nmol·mg protein^-1·min^-1 (P < 0.001). K(m) values were 0.017 ± 0.001 μM, which was not significantly different from control values. Basolateral membranes depleted of calmodulin by EDTA osmotic shock decreased ATP-dependent Ca²⁺ uptake by 65%. Trifluoperazine, an anticalmodulin drug, inhibited ATP-dependent Ca²⁺ uptake by 50%, while no inhibition was noted in calmodulin-depleted membranes. Efflux of Ca²⁺ in the BLMVs was stimulated by trans-Na⁺. Na⁺-dependent Ca²⁺ uptake was saturable with respect to Ca²⁺ concentration and exhibited a KM of 0.09 ± 0.03 μM and a V(max) of 1.08 ± 0.01 nmol·mg protein^-1·min^-1. These results are consistent with the existence of a Na⁺-Ca²⁺ exchange system and ATP and Mg²⁺-dependent, calmodulin-regulated Ca²⁺, transport mechanism in BLMVs of human enterocytes.