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Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles
Published on: December 31, 2019
Divalent cation transport by vesicular nucleotide transporter
Takaaki Miyaji1, Keisuke Sawada, Hiroshi Omote
1Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan.
The Journal of Biological Chemistry
|November 5, 2011
Summary
The vesicular nucleotide transporter (VNUT) imports divalent cations like Mg(2+) and Ca(2+) into secretory vesicles, likely as chelating complexes with ATP. This suggests VNUT functions as a divalent cation importer under physiological conditions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The vesicular nucleotide transporter (VNUT) facilitates ATP storage and exocytosis from secretory vesicles.
- VNUT's ATP transport is membrane potential-dependent and independent of divalent cations.
- The transport of divalent cation-ATP complexes by VNUT remains uncharacterized.
Purpose of the Study:
- To investigate whether VNUT transports divalent cations, such as Mg(2+) and Ca(2+).
- To elucidate the mechanism and physiological role of VNUT in divalent cation transport.
Main Methods:
- Utilized proteoliposomes reconstituted with purified VNUT.
- Measured Mg(2+) uptake via atomic absorption spectroscopy.
- Assessed radioactive Ca(2+) uptake under varying membrane potential and pH conditions.
- Employed site-directed mutagenesis (Arg-119 to Alanine) and RNA interference (RNAi).
Main Results:
- VNUT-containing proteoliposomes actively transported Mg(2+) and Ca(2+) in an ATP- and membrane potential-dependent manner.
- Ca(2+) uptake was inhibited by Evans blue and various divalent cations, and required millimolar Cl(-).
- Mutating Arg-119 abolished both ATP and Ca(2+) transport.
- RNAi knockdown of VNUT reduced vesicular Mg(2+) concentration by 32.3%.
Conclusions:
- VNUT transports both nucleotides and divalent cations, likely as chelating complexes.
- VNUT functions as a divalent cation importer in secretory vesicles.
- These findings reveal a novel role for VNUT in regulating intracellular divalent cation concentrations.
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