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

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.

FIGURE 1.

Syntheses of R-LA, S-LA, and lipoamides. A, chemical scheme to prepare R-LA and S-LA. R-LA and S-LA were isolated by classical resolution of twice recrystallized R,S-LA by reaction with RAMBA or SAMBA followed by multiple recrystallizations of the diastereomeric salt pairs R-LA-RAMBA and S-LA-SAMBA followed by liberation of the free, enantiomeric salt pairs with dilute acid and recrystallization for >95% R-LA or S-LA. To increase the enantiomeric excess, the method was repeated using the opposite resolving agent and seeded with preformed crystals of the undesired salt pair. The hydrocarbon solutions were extracted with aqueous acid to remove excess chiral amine, washed with H₂O, dried, evaporated, and recrystallized, yielding essentially enantiomerically pure (>98%) crystals of R-LA or S-LA. B, chemical scheme to prepare R,S-LAM, R-LAM, and S-LAM. The enantiomerically pure acids were used to make the amides via the mixed anhydride method (19). LA was dissolved in THF and incubated with equimolar triethylamine and ethyl chloroformate. The intermediate mixed anhydride was incubated with 28% aqueous ammonia.

FIGURE 2.

Analysis of hSMVT-mediated uptake in oocytes. A, uptake of R,S-[³H]LA (1 Ci/mmol) in hSMVT-expressing oocytes (squares) or non-injected control oocytes (circles) in the presence of 100 mm NaCl (solid symbols) or choline chloride (open symbols). B, uptake of 1.66 μm R-[³H]LA (4 Ci/mmol) was measured for 5 min in hSMVT-expressing (hSMVT) or non-injected control (co) oocytes in the presence of 100 mm choline chloride (open) or NaCl (solid). C, time course of 1.66 μm R-[³H]LA (4 Ci/mmol) uptake by hSMVT-injected oocytes (■; n = 10) performed in the presence of 100 mm NaCl. Non-injected oocytes (○; n = 11) served as control. D, kinetics of R-[³H]LA uptake in hSMVT-expressing oocytes. Uptake of R-[³H]LA (1 Ci/mmol) was measured for 1-min intervals in hSMVT-expressing oocytes (n = 8) with R-[³H]LA concentrations varying from 0.2 to 53.12 μm. Fitting the data to the Michaelis-Menten equation yielded a K_m of 4.0 ± 0.5 μm and V_max of 15.5 ± 0.6 pmol of R-[³H]LA × oocyte⁻¹ × min⁻¹. E, ion dependence of hSMVT expressed in oocytes. Uptake of 1.66 μm R-[³H]LA in hSMVT-expressing oocytes (solid bars) or control oocytes (open bars) measured for 15 min in the presence of 100 mm concentrations of either choline-chloride (CHO) at pH 7.4 or 5.5, NaCl, sodium gluconate (NaG), or LiCl. F, effect of stereoisomers of LA and lipoamide on the transport of R-[³H]LA. Uptake of 1.66 μm R-[³H]LA was measured for 30 min in the presence of 50 μm concentrations of the indicated compounds in control (open bars; n = 8) and hSMVT-expressing (solid bars; n = 9) oocytes (Bio, biotin; PA, pantothenic acid). Data are from representative experiments (repeated in triplicate with different batches of oocytes) and are shown as the mean ± S.E. (error bars) of n determinations. Kinetic constants in D were obtained by fitting the data to the Michaelis-Menten non-linear regression fitting in GraphPad Prism 5 and are shown as the mean ± S.E. (error bars) of the fit.

FIGURE 3.

Purification of recombinant hSMVT produced in P. pastoris. A, time course of 1.66 μm R-[³H]LA (4 Ci/mmol) uptake by oocytes injected with mRNA encoding the native (■) or recombinant (FLAG epitope and His tag-containing) (▿) hSMVT or by water-injected oocytes (○) in the presence of 100 mm NaCl. Data are from a representative experiment repeated three times with oocytes from different batches. Error bars represent S.E. of multiple determinations (n ≥ 6). B and C, immunological detection of the recombinant hSMVT (hS) in membranes of P. pastoris with monoclonal anti-FLAG M2 IgG after subjecting samples to 11% SDS-PAGE. B, membranes from P. pastoris cells harboring a control plasmid devoid of the recombinant hSMVT gene served as control (co). C, recombinant hSMVT in P. pastoris membrane vesicles (−) was subjected to peptidyl N-glycosidase F treatment (indicated by “+”). Protein standards are shown between B and C. D, purification of hSMVT. Membranes were solubilized with 1.5% DDM (Sol), and hSMVT was bound to the α-FLAG affinity resin, washed (Wa), and eluted by competition with the FLAG peptide (El). 2 μg of purified hSMVT (El) and samples with comparable fractional protein amount were subjected to 14% SDS-PAGE followed by silver staining.

FIGURE 4.

Interaction of R-LA with purified hSMVT. Binding of R-[³H]LA to 50 ng of purified hSMVT was measured with the SPA by immobilizing recombinant hSMVT via the engineered His tag to copper-coated yttrium silicate SPA beads. A, binding of 0.83 μm R-[³H]LA (12 Ci/mmol) was assayed in the presence of 150 mm NaCl or Tris/Mes. To determine the nonspecific background binding activity, 800 mm imidazole (imid) was added to the samples because imidazole competes with the His tag of recombinant hSMVT for binding to the copper-coated SPA beads. B, effect of stereoisomers of LA and derivatives on R-[³H]LA equilibrium binding. Binding of 0.4 μm R-[³H]LA was tested in 150 mm NaCl in the presence or absence of the indicated compounds at 50 μm. C, saturation binding of R-[³H]LA (0.1 Ci/mmol) to hSMVT in the presence of 150 mm NaCl. Saturation equilibrium binding was performed with increasing concentrations (0.2–25 μm) of R-[³H]LA. Data fits of the two phases were performed independently with one-site models. Fitting the data ranging between 0 and 5 μm R-[³H]LA yielded a K_d of 0.9 ± 0.03 μm with a Hill coefficient of 1.99 ± 0.11, and fitting the data points between 5 and 25 μm R-[³H]LA yielded a K_d of 7.38 ± 1.44 μm. The B_max was calculated to be 1.84 ± 0.09 molecules of LA bound per molecule of hSMVT. Kinetic constants represent the mean ± S.E. of the fit. D, silver-stained SDS-polyacrylamide gel of hSMVT-containing nanodiscs. hSMVT was assembled into nanodiscs of different lipid composition: E. coli polar lipid (ND1), E. coli polar lipid with 6% (ND2) or 10% cholesterol (ND3), and POPC/POPG (3:2) with 6% cholesterol (ND4). After reconstitution, samples of the nanodiscs preparations (ND) and the supernatant (sup) that correspond to the amount of hSMVT (hS) and the membrane scaffold protein (MSP) used for the reconstitution were analyzed by 14% SDS-PAGE. For comparison, the amount of purified MSP1E3D1 (lane MSP; open arrowhead) and hSMVT (lane hS; solid arrowhead) used for the reconstitution are shown. E, comparison of the binding activity of hSMVT in different lipid environments. Binding of 20 μm R-[³H]LA (0.1 Ci/mmol) was assessed with the SPA using 50 ng of hSMVT in purified, detergent-solubilized form or reconstituted into nanodiscs of different lipid composition (ND1–4; see D for details). hSMVT amounts in nanodiscs were calculated based on densitometry measurements in NIH ImageJ of hSMVT-containing nanodiscs subjected to SDS-PAGE followed by silver staining. Data in A, B, C, and E are from representatives experiments performed ≥2 times and are shown as mean ± S.E. (error bars) of triplicate determinations.

FIGURE 5.

Activity of hSMVT in proteoliposomes. Affinity chromatography-purified hSMVT was reconstituted into preformed liposomes made of E. coli lipids containing 10% (w/w) cholesterol. A, silver-stained SDS-PAGE of hSMVT-containing proteoliposomes. Proteoliposomes were incubated with Ni²⁺ affinity resin to capture the proteoliposome fraction with externally located His tag. The flow-through (Fl), washed (Wa), and imidazole-eluted (El) fractions were collected and applied on the gel. Proteoliposomes (PL) not subjected to the affinity chromatography are shown for comparison. B, time course of 0.83 μm R-[³H]LA uptake in hSMVT-containing proteoliposomes in the presence (■) or absence (▾) of 100 mm NaCl. Empty liposomes served as control (○). C, kinetics of R-[³H]LA uptake in hSMVT-containing proteoliposomes. Uptake of R-[³H]LA (1 Ci/mmol) was measured for 1-min intervals with R-[³H]LA concentrations varying from 0.2 to 53.12 μm. Fitting the data to the Michaelis-Menten equation yielded a K_m of 3.9 ± 0.6 μm and a V_max of 0.98 ± 0.04 μmol of R-[³H]LA × mg of hSMVT⁻¹ × min⁻¹, resulting in a k_cat of transport of 1.13 ± 0.05 s⁻¹. Data in B and C are from representative experiments (repeated ≥2 times) and are shown as the mean ± S.E. (error bars) of triplicate determinations. Kinetic constants are shown as the mean ± S.E. (error bars) of the fit.