Calcium/calmodulin inhibition of coupled NaCl transport in membrane vesicles from rabbit ileal brush border

The role of Ca²⁺ and calmodulin in regulating coupled NaCl transport has been investigated in membrane vesicles from rabbit ileal brush border. Uptake of ²²Na⁺ and ³⁶Cl^- was determined by a rapid filtration technique in vesicles isolated with a sucrose density gradient ultracentrifugation method. Ca²⁺ on the inside of the vesicle inhibited Na⁺ uptake when Cl^- was the anion and Cl^- uptake when Na⁺ was the cation by ~30%. Ca²⁺ on the outside had no effect. When gluconate was the anion or when choline was the cation, Na⁺ or Cl^- uptake was reduced by only 9-12%. A similar inhibition of D-[³H]mannitol uptake (10-17%) suggests this was due to a nonspecific decrease in the membrane permeability. Other cations such as Ba²⁺ and Mg²⁺ had no effect, but La³⁺ inhibited Na⁺ and Cl^- uptake to the same degree as Ca²⁺. Calmodulin (2 μM) in combination with Ca²⁺ (1 μM, free concentration) significantly inhibited Na⁺ uptake when Cl^- was the anion by 21-32% and Cl^- uptake when Na⁺ was the cation by 20-27%. This effect was completely reversed by 10 μM trifluoperazine. When gluconate was the anion or when choline was the cation, Na⁺ or Cl^- uptake was unaffected by Ca²⁺/calmodulin and trifluoperazine. The K(i) for Ca²⁺ inhibition of Cl^--coupled Na⁺ uptake was reduced from 200 μM to 0.2 μM by incubation with 20 μM calmodulin. The K(i) for exogenously added calmodulin studied at 1 μM Ca²⁺ was 0.2 μM. The K(i) for trifluoperazine inhibition of the Ca²⁺/calmodulin response was 3 μM. These results represent compelling evidence for intracellular Ca²⁺/calmodulin regulation of coupled NaCl transport across the intestinal microvillus membrane. The exact mechanism of this regulation remains to be delineated.