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

Abstract

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 approximately equal to 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 microM) in combination with Ca2+ (1 microM, 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 microM trifluoperazine. When gluconate was the anion or when choline was the cation, Na+ or Cl- uptake was unaffected by Ca2+/calmodulin and trifluoperazine. The Ki for Ca2+ inhibition of Cl- -coupled Na+ uptake was reduced from 200 microM to 0.2 microM by incubation with 20 microM calmodulin. The Ki for exogenously added calmodulin studied at 1 microM Ca2+ was 0.2 microM. The Ki for trifluoperazine inhibition of the Ca2+/calmodulin response was 3 microM. 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.