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A novel method for the efficient entrapment of calcium in large unilamellar phospholipid vesicles

Authors
Journal
Biochimica et Biophysica Acta (BBA) - Biomembranes
0005-2736
Publisher
Elsevier
Publication Date
Volume
1025
Issue
1
Identifiers
DOI: 10.1016/0005-2736(90)90197-v
Keywords
  • Ionophore A23187
  • Transmembrane Ph Gradient
  • Ph Gradient
  • Phosphatidylcholine Vesicle
  • Large Unilamellar Vesicle
  • Cation Uptake

Abstract

Abstract A technique for the efficient entrapment of high concentrations of Ca 2+ in large unilamellar phospholipid vesicles (LUVs), using the carboxylic acid antibiotic ionophore A23187 (calcimycin) is demonstrated. It is shown that rapid A23187-mediated entrapment of Ca 2+, corresponding to essentially 100% sequestration of the extravesicular cation may be achieved for egg yolk phosphatidylcholine LUVs (100 nm) in the presence of a transmembrane proton gradient (acidic interior). Interior-exterior concentration cation gradients of over 400-fold may be readily achieved, with interior Ca 2+ concentrations in excess of 250 mM. It is shown that the extent and efficiency of the A23187-mediated uptake process is affected by the intravesicular buffering capacity and the extravesicular Ca 2+ concentration in a manner that is consistent with a Ca 2+-H + exchange process. In the absence of a pH gradient, or the presence of a reversed gradient (basic interior), only background levels of cation uptake are detected. The driving force for A23187-mediated uptake of Ca 2+ is shown to depend on the intravesicular proton pool rather than on a chelation process. This protocol provides a novel method for the efficient entrapment of high concentrations of Ca 2+ and other cations in phospholipid vesicles.

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