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

C. C. Fan, D. W. Powell

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 ~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.

Original languageEnglish (US)
Pages (from-to)5248-5252
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume80
Issue number17 I
StatePublished - 1983
Externally publishedYes

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Calmodulin
Microvilli
Rabbits
Calcium
Cations
Membranes
Trifluoperazine
Anions
Choline
Ultracentrifugation
Mannitol
Sucrose
Permeability

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Calcium/calmodulin inhibition of coupled NaCl transport in membrane vesicles from rabbit ileal brush border. / Fan, C. C.; Powell, D. W.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, No. 17 I, 1983, p. 5248-5252.

Research output: Contribution to journalArticle

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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 ~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.",
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