The sodium/multivitamin transporter: a multipotent system with therapeutic implications

Abstract

The Na(+)/multivitamin transporter (SMVT) is a member of the solute:sodium symporter family that catalyzes the Na(+)-dependent uptake of the structurally diverse water-soluble vitamins pantothenic acid (vitamin B5) and biotin (vitamin H), α-lipoic acid-a vitamin-like substance with strong antioxidant properties-and iodide. The organic substrates of SMVT play central roles in the cellular metabolism and are, therefore, essential for normal human health and development. For example, biotin deficiency leads to growth retardation, dermatological disorders, and neurological disorders. Animal studies have shown that biotin deficiency during pregnancy is directly correlated to embryonic growth retardation, congenital malformation, and death of the embryo. This chapter focuses on the structural and functional features of the human isoform of SMVT (hSMVT); the discovery of which was greatly facilitated by the cloning and expression of hSMVT in tractable expression systems. Special emphasis will be given to mechanistic implications of the transport process of hSMVT that will inform our understanding of the molecular determinants of hSMVT-mediated transport in dynamic context to alleviate the development and optimization of hSMVT as a multipotent platform for drug delivery.

Keywords: Biotin; Lipoic acid; Na(+)-coupled transport; Pantothenic acid; SLC5; Sodium/multivitamin transporter; Solute:sodium symporter family.

Figure 1. Kinetic model for the SSS member SGLT1

A six-state kinetic model of the hSGLT1 transport cycle was proposed (Parent et al., 1992b; Wright et al., 1994) according to which 2 external Na⁺ bind to the empty, outward-facing transporter [T]_o. The Na⁺-bound transporter [TNa₂]_o is poised to bind sugar (S), yielding the Na⁺ and sugar-bound intermediate [TNa₂S]_o. Under physiological conditions this conformational state changes its accessibility to the inward-facing conformation [TNa₂S]_i which is followed by the internal sequential release of sugar ([TNa₂]_i) and Na⁺ ([T]_i). However, all state transitions are reversible. Since the transient charge relaxations are eliminated by transported sugars (compare Fig. 9B, left panel for hSGLT1), only the three shaded conformational states participate effectively in the observed charge transfer. Membrane voltage affects Na⁺ binding and the transition between [T]_o↔[T]_i (Loo, Hirayama, Cha, Bezanilla, & Wright, 2005). In the absence of substrate, the uncoupled flux of Na⁺ (‘Na⁺-leak’) is observed, which is ~ 10 % of the maximum sugar-induced currents (Loo et al., 1993). ¹⁴C-sugar and ²²Na⁺ flux studies show the direct correlation between the fluxes of substrates and charge (Mackenzie, Harper, Taylor, & Rennie, 1994).

Figure 2. Deduced topology of hSMVT

The secondary structure of hSMVT is based on hydrophobicity calculations (Kyte & Doolittle, 1982) and contains 12 transmembrane domains connected by hydrophilic extracellular and intracellular loops. N and C termini are facing in the cytoplasm. Putative N-glycosylation sites are highlighted (bold) and the two putative PKC phosphorylation sites are shown as rectangles(Wang et al., 1999)s.

Figure 3. Characterization of the hSMVT protein

A) Immunological detection of native hSMVT using a polyclonal antibody against SMVT (taken from de Carvalho & Quick (de Carvalho & Quick, 2011)). Non-injected oocytes (co) served as control. B) hSMVT was subjected to peptidyl N-glycosidase F treatment (indicated by “+”) to show the glycosylation of hSMVT (M.Q. unpublished).

Figure 4. The phylogenetic tree of the SSS family

The tree was generated by the workflow integrated in the phylogeny.fr server (Dereeper et al., 2008). Briefly, the sequences of selected SSS members were aligned by the MUSCLE program (Edgar, 2004) and curated by the Gblock program (Castresana, 2000); the tree was constructed with the maximum likelihood method using PhyML (Guindon & Gascuel, 2003) and rendered by TreeDyn (Chevenet, Brun, Banuls, Jacq, & Christen, 2006). The SSS members are indicated by their Swissprot codes and commonly known abbreviations. For simplicity, the lengths of the branches were not indicated.

Figure 5. Predicted topology of hSMVT

A) Pair-wise sequence alignment between hSMVT and vSGLT extracted from a structure-based and manually-adjusted multiple sequence alignment of all the available non-redundant prokaryotic and eukaryotic SSS sequences from refseq database (>1000 sequences). Identical residues in vSGLT and hSMVT are highlighted and the transmembrane domains in vSGLT (Faham et al., 2008) are indicated. B) The diagram of hSMVT secondary structure generated by the RbDe program (Skrabanek, Campagne, & Weinstein, 2003), based on the pair-wise sequence alignment in A.

Figure 6. The structures of the organic substrates of hSMVT

A) biotin, B) pantothenic acid, C) (oxidized) R-α-lipoic acid, and D) R-dihydrolipoic acid.

Figure 7. Substrate transport by hSMVT in oocytes

A) Uptake of 1.4 µM ¹⁴C-pantothenic acid (PA), 2.2 µM ¹⁴C-biotin (BIO), 1.6 µM ³H- R-α-lipoic acid (LA) or 500 µM K[¹²⁵I⁻] was measured for 15 min in hSMVT-expressing or control (co) oocytes in 100 mM choline chloride (open) or NaCl (solid). B) Substrate-elicited inward currents in a hSMVT-expressing oocyte at a holding potential of −50 mV in 100 mM NaCl. 25 µM BIO, PA, LA, or NaI (I⁻) were added as indicated.

Figure 8. hSMVT-mediated ionic currents

A) Current-voltage (I/V) relationship of total steady-state currents in hSMVT-expressing oocytes upon stepping the holding membrane potential of −50 mV to a series of test voltages (V_t) from +50 to −150 mV in the presence of 100 mM choline chloride, pH 7.4 (●) or pH 5.5 (▼), 100 mM NaCl=Na-gluconate (O) or 100 mM LiCl (∆), at pH 7.4. hSMVT exhibited no detectable Na⁺ leak currents under our experimental conditions (de Carvalho & Quick, 2011). For example, these currents have been observed for NIS (Eskandari et al., 1997) and SGLT1 (Hirayama et al., 1997) when 100 mM choline chloride was replaced with 100 mM NaCl in assay buffer at pH 7.4. Also note that substrate-elicited currents were only observed in the presence of Na⁺B) Current-voltage (I/V) relationship of substrate-induced steady-state currents elicited in hSMVT-expressing oocytes upon stepping the holding membrane potential of −50 mV to a series of test voltages (V_t) from +50 to −150 mV. 25 µM PA, BIO, LA, or KI (same abbreviations as in Figure 7) were used were added to buffer containing 100 mM NaCl. C) I/V relationship of currents induced by varying concentrations of PA in 100 mM NaCl. PA-induced currents were plotted as a function of [PA] for each V_t (except −10 and −30 mV), yielding D) the maximum current (I_max) and E) the apparent affinity (EC₅₀) of PA-induced electrical currents.

Figure 9. Current recordings in Xenopus laevis ooctes injected with hSGLT1- or hSMVT-cRNA in response to step changes in the test voltage

The test membrane potential (V_t) was stepped from a holding potential (V_h) of −50 mV to V_t of +50 to −150 mV in 20 mV decrements for 100 ms (on currents) before returning to V_h (off currents). A) Currents were recorded in the presence of 100 mM external NaCl in individual hSGLT-, hSMVT, or water-injected control (co) oocytes. B) Currents were recorded the same oocytes shown in A) after the addition of 5 mM α-methyl-D-glucopyranoside (αMDG; hSGLT1, left panel) or 10 µM LA (hSMVT, right panel). The addition of 5 mM α MDG or 10 µM LA did not affect the current response in control oocytes (not shown). C) Charge transfer. The charge-voltage (Q–V) relationship for hSGLT1 (top panel) and hSMVT (lower panel) in the presence of 100 mM NaCl (corresponding to the left and center panel in A) were calculated according to the Boltzmann equation.