Surprising substrate versatility in SLC5A6: Na+-coupled I- transport by the human Na+/multivitamin transporter (hSMVT)

Abstract

Iodide (I(-)) is an essential constituent of the thyroid hormones triiodothyronine and thyroxine, which are required for the development of the central nervous system in the fetus and newborn. I(-) uptake in the thyroid is mediated by the Na(+)/I(-) symporter (NIS). NIS has gained particular medical interest due to its sensitivity to the environmental pollutant perchlorate (ClO(4)(-)) and its implication in radioiodide cancer treatment. Recently, others have shown that I(-) absorption in the intestine is mediated by NIS (Nicola, J. P., Basquin, C., Portulano, C., Reyna-Neyra, A., Paroder, M., and Carrasco, N. (2009) Am. J. Physiol. Cell Physiol. 296, C654-662). However, their data suggest the participation of other systems in the homeostasis of I(-), in particular because in vivo uptake studies revealed a ClO(4)(-)-insensitive transport component. Here, we describe Na(+)-coupled I(-) uptake by the human Na(+)/multivitamin transporter (hSMVT), a related protein isolated from the placenta, where it was suggested to supply the fetus with the water-soluble vitamins biotin and pantothenic acid, and α-lipoic acid. hSMVT-mediated Na(+)/I(-) symport is inhibited by the other three organic hSMVT substrates but not by NIS substrates; notably, hSMVT is insensitive to ClO(4)(-). Because hSMVT is found in the intestine and in many other tissues, we propose that hSMVT may play an important role in the homeostasis of I(-) in the body.

FIGURE 1.

Western blot analysis of oocytes expressing hSMVT. Immunological detection of hSMVT was performed using a polyclonal antibody raised against a 22-amino acid peptide corresponding to positions 44–65 in hSMVT as described under “Experimental Procedures.” Protein originating from three oocytes (co, water-injected control oocytes; hSMVT, hSMVT-expressing oocytes) was subjected to 10% SDS-PAGE and electroblotted onto a PVDF transfer membrane. The membrane was incubated with rabbit anti-hSMVT polyclonal IgG, followed by incubation with goat anti-(rabbit IgG) horseradish peroxidase conjugate. Immunoreactions were visualized with the enhanced chemiluminescence method. Positions of the protein standards are indicated (kDa).

FIGURE 2.

Transport activity of hSMVT in Xenopus oocytes. A, uptake of 25 μm d-[¹⁴C]biotin by oocytes injected with hSMVT mRNA. Transport of radiolabeled biotin was assayed for 15 min at 25 °C in the presence of 100 mm choline chloride, pH 7.4 (CHO), 100 mm NaCl (Na⁺) plus 250 μm of the indicated compounds: PA, RS-α-lipoic acid (LA), or NaI (I⁻). B, uptake of 50 μm RS-[³H]α-lipoic acid by hSMVT-expressing (solid bars) and control (open bars) oocytes. Assays were performed as described in the legend to A by including Cl⁻-free conditions; 100 mm NaCl were equimolarly replaced with sodium gluconate (NaG). Compounds, biotin (BIO), PA, or I⁻ were added at a final concentration of 100 μm. C, the inhibition of LA uptake is dependent on the concentration of I⁻. Uptake of RS-[³H]α-lipoic acid was performed in 100 mm NaCl in the presence of increasing [KI]. Data were plotted as function of I⁻ concentration and fitted according to a modified Equation 1 (n set to 1; see text for details). Data are from representative experiments.

FIGURE 3.

Substrate-elicited inward currents of hSMVT. Currents were recorded from a representative oocyte injected with hSMVT mRNA at V_h = −50 mV in 100 mm NaCl-containing buffer. 25 μm biotin (BIO), PA, RS-α-lipoic acid (LA), or NaI (I⁻) were added as indicated by the bar.

FIGURE 4.

hSMVT-elicited electrical currents. A, current-voltage (I/V) relationship of steady-state currents elicited in hSMVT-expressing oocytes in the presence of different ions upon stepping the membrane potential from V_h of −50 mV to a series of test voltages (V_t) from +50 to −150 mV, in 20-mV increments. The relevant ionic condition of the assay buffer is indicated: 100 mm choline chloride, pH 7.4 or 5.5 (CHO, pH 7.4 or CHO, pH 5.5, respectively); 100 mm NaCl or sodium gluconate (NaG), or LiCl. B, substrate-induced currents. 25 μm PA, biotin, or LA were added to assay buffer containing 100 mm NaCl, and currents were recorded as described in the legend to A. The substrate-induced currents were calculated by subtracting the currents observed in 100 mm NaCl from the total current in NaCl plus the indicated substrate. C, effect of representative potential substrates (1 mm isethionate, 1 mm perchlorate [ClO₄⁻], 100 mm α-methyl-d-glucose (α-MDG) on hSMVT-mediated steady-state currents in 100 mm NaCl. I/V relations of the net currents (I^compound = I^total − I^{no compound}) are shown. D, I/V relation of I⁻-induced steady-state currents in 100 mm NaCl and increasing concentrations of [I⁻]. I⁻-induced currents were plotted as a function of [I⁻] for each V_t from −150 to +10 mV and fitted to Equation 1 with n = 1, yielding K_0.5^I− (E) and I_max^I− (F) at each indicated V_t.

FIGURE 5.

Iodide transport by hSMVT. Uptake of 500 μm ¹²⁵I⁻ was measured in oocytes injected with hSMVT mRNA or in control oocytes. A, cation dependence of ¹²⁵I⁻ uptake. Transport was measured for 5 min in the presence of the indicated assay buffer (100 mm choline chloride, pH 7.4 (−), or pH 5.5 (Δμ_H⁺), 100 mm LiCl ((Δμ_Li⁺), 100 mm NaCl (Δμ_Na⁺/Δμ_Cl⁻) or 100 mm sodium gluconate (Δμ_Na⁺). B, time course of Na⁺-dependent I⁻ uptake in hSMVT-expressing (■) and control (○) oocytes. C, inhibition of ¹²⁵I⁻ transport by hSMVT substrates. D, isotopic dilution of 500 μm ¹²⁵I⁻ with nonradioactive I⁻ yielding an IC₅₀^I− of 267.1 ± 39.5 μm. E, Na⁺ dependence of the initial rates of ¹²⁵I⁻ transport by hSMVT. Transport of 500 μm ¹²⁵I⁻ was measured at increasing concentrations of sodium gluconate for 5 min.