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J Biol Chem. 2015 Jun 26;290(26):16372-82. doi: 10.1074/jbc.M114.622555. Epub 2015 May 13.

Interaction of α-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT).

Author information

1
From the Center for Molecular Recognition and.
2
Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032.
3
GeroNova Research, Inc., Richmond, California 94806, and david@geronova.com.
4
From the Center for Molecular Recognition and Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032 mq2102@cumc.columbia.edu.

Abstract

The human Na(+)/multivitamin transporter (hSMVT) has been suggested to transport α-lipoic acid (LA), a potent antioxidant and anti-inflammatory agent used in therapeutic applications, e.g. in the treatment of diabetic neuropathy and Alzheimer disease. However, the molecular basis of the cellular delivery of LA and in particular the stereospecificity of the transport process are not well understood. Here, we expressed recombinant hSMVT in Pichia pastoris and used affinity chromatography to purify the detergent-solubilized protein followed by reconstitution of hSMVT in lipid bilayers. Using a combined approach encompassing radiolabeled LA transport and equilibrium binding studies in conjunction with the stabilized R-(+)- and S-(-)-enantiomers and the R,S-(+/-) racemic mixture of LA or lipoamide, we identified the biologically active form of LA, R-LA, to be the physiological substrate of hSMVT. Interaction of R-LA with hSMVT is strictly dependent on Na(+). Under equilibrium conditions, hSMVT can simultaneously bind ~2 molecules of R-LA in a biphasic binding isotherm with dissociation constants (Kd) of 0.9 and 7.4 μm. Transport of R-LA in the oocyte and reconstituted system is exclusively dependent on Na(+) and exhibits an affinity of ~3 μm. Measuring transport with known amounts of protein in proteoliposomes containing hSMVT in outside-out orientation yielded a catalytic turnover number (kcat) of about 1 s(-1), a value that is well in agreement with other Na(+)-coupled transporters. Our data suggest that hSMVT-mediated transport is highly specific for R-LA at our tested concentration range, a finding with wide ramifications for the use of LA in therapeutic applications.

KEYWORDS:

SLC5; Xenopus; kinetics; lipoamide; lipoic acid; membrane transport; scintillation proximity assay; solute/sodium symporter; stereoselectivity; yeast

PMID:
25971966
PMCID:
PMC4481234
DOI:
10.1074/jbc.M114.622555
[Indexed for MEDLINE]
Free PMC Article

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