Molecular cloning of the mouse IMINO system: an Na+- and Cl--dependent proline transporter

Abstract

Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of the long-sought IMINO system. The mouse has two homologues corresponding to the single human SLC6A20 gene: these have been named XT3 and XT3s1. Expression of mouse XT3s1, but not XT3, in Xenopus laevis oocytes induced an electrogenic Na+-and-Cl--dependent transporter for proline, hydroxyproline, betaine, N-methylaminoisobutyric acid and pipecolic acid. Expression of XT3s1 was found in brain, kidney, small intestine, thymus, spleen and lung, whereas XT3 prevailed in kidney and lung. Accordingly we suggest that the two homologues be termed 'XT3s1 IMINO(B)' and 'XT3 IMINO(K)' to indicate the tissue expression of the two genes.

Figure 1. Expression of XT3 and XT3s1 in mouse tissues

Total RNA was isolated from different mouse tissues and reverse-transcribed into cDNA. Specific fragments were amplified for XT3 (A), XT3s1 (B) and for actin (C). Tissues are labelled as follows: K, kidney; Li, liver; Si, small intestine; Cor, brain cortex; Cer, brain cerebellum; Bst, brain stem; Thy, thymus; Sp, spleen; H, heart; Lu, lung; M, muscle. Markers were loaded on each side of the gel. The arrows in all three panels point to standards of 1000 and 1500 bp in length. (D) In situ hybridization of XT3/XT3s1 in mouse tissues. The antisense probe was hybridized to mRNA in sections from brain (Br), small intestine (Si), kidney (Ki). In the brain the highest expression was found in the pia mater (arrow, panel Br1) and the plexus choroideus (arrow, panel Br2). In the kidney, XT3/XT3s1 mRNA was detected throughout the cortex. In the small intestine mRNA localized in villus enterocytes, with the highest expression in basal cells (arrows).

Figure 2. Substrate specificity of XT3s1

Oocytes were injected with XT3 or XT3s1 cRNA or remained uninjected (n.i.) in the controls. (A) Uptake of [¹⁴C]glutamine, [¹⁴C]glutamate, [¹⁴C]leucine, [¹⁴C]histidine, [¹⁴C]alanine, [¹⁴C]phenylalanine, [¹⁴C]glycine, [¹⁴C]proline and [¹⁴C]arginine (100 μM each) was determined 4 days after injection in oocytes expressing XT3 (grey bars), XT3s1 (black bars) and in non-injected oocytes (white bars). (B) Uptake of 50 μM [¹⁴C]proline was challenged by 5 mM unlabelled amino acids and their analogues. Each bar represents the transport activity (mean±S.D.) for ten oocytes (e=3). Abbreviations: BCH, 2-aminobicyclo[2.2.1]-heptane-2-carboxylic acid; Bet, betaine. (C) Original tracings of substrate-induced currents (0.5 mM) in XT3s1-expressing oocytes after 4 days incubation. Superfusion intervals are indicated by bars. Abbreviation: HO-Pro, hydroxyproline. (D) Summary of substrate-induced inward currents. Substrate-induced currents (0.5 mM) were compiled from experiments of the type shown in (C) and normalized to proline-induced currents. The experiment was performed with seven oocytes (e=3). Noninjected oocytes showed inward currents of 2–3 nA in response to the same panel of substrates. Abbreviations: Nip, nipecotic acid; Pip, pipecolic acid.

Figure 3. Ion activation of proline transport by XT3s1

Oocytes were injected with XT3s1 cRNA or remained uninjected (n.i.) in the controls. (A) [¹⁴C]Proline (100 μM) uptake was determined 4 days after injection in buffer containing NaCl or in buffer where NaCl was replaced by LiCl, NMDG-Cl or sodium gluconate. Each bar represents the transport activity (mean±S.D.) for ten oocytes (e=3). (B) Proline-induced currents (5 mM) plotted as a function of the extracellular Na⁺ concentration. (C) Proline-induced currents plotted as a function of the extracellular Cl⁻ concentration. Curves display the transport activity (mean±S.D.) for seven oocytes (e=3).