Interaction of {alpha}-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT) [Bioenergetics]

May 13th, 2015 by Zehnpfennig, B., Wiriyasermkul, P., Carlson, D. A., Quick, M.

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′s Disease. However, the molecular basis of the cellular delivery of LA and, in particular, the stereospecificity of the transport process is 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 RS-(+/-) racemic mixture of LA or lipoamide (LAM) we identified the biological 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 ~ two molecules of R-LA in a biphasic binding isotherm with dissociation constants (kd) of 0.9 μM 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.