Human solute carrier SLC6A14 is the beta-alanine carrier

Abstract

The beta-alanine carrier was characterized functionally in the 1960s to 1980s at the luminal surface of the ileal mucosal wall and is a Na(+)- and Cl(-)-dependent transporter of a number of essential and non-essential cationic and dipolar amino acids including lysine, arginine and leucine. beta-Alanine carrier-like function has not been demonstrated by any solute carrier transport system identified at the molecular level. A series of experiments were designed to determine whether solute carrier SLC6A14 is the molecular correlate of the intestinal beta-alanine carrier, perhaps the last of the classical intestinal amino acid transport systems to be identified at the molecular level. Following expression of the human SLC6A14 transporter in Xenopus laevis oocytes, the key functional characteristics of the beta-alanine carrier, identified previously in situ in ileum, were demonstrated for the first time. The transport system is both Na(+) and Cl(-) dependent, can transport non-alpha-amino acids such as beta-alanine with low affinity, and has a higher affinity for dipolar and cationic amino acids such as leucine and lysine. N-methylation of its substrates reduces the affinity for transport. These observations confirm the hypothesis that the SLC6A14 gene encodes the transport protein known as the beta-alanine carrier which, due to its broad substrate specificity, is likely to play an important role in absorption of essential nutrients and drugs in the distal regions of the human gastrointestinal tract.

Figure 1. Amino acid uptake in SLC6A14-injected and water-injected oocytes

Uptake of various radiolabelled amino acids (2–5 μCi ml⁻¹; all 2 μm except [¹⁴C]MeAIB which was 20 μm) was measured over 40 min in a NaCl-containing pH 7.4 solution into oocytes injected with either SLC6A14 cRNA or water (n= 18–20). NS, P > 0.05; **P < 0.01; ***P < 0.001 all versus water-injected oocytes.

Figure 2. Na⁺- and Cl⁻-dependent β-alanine uptake by SLC6A14

[³H]β-Alanine uptake (2 μm) was measured under control conditions (NaCl-containing solution, pH 7.4), in Na⁺-free pH 7.4 (–Na⁺) solution, in Cl⁻-free pH 7.4 (–Cl⁻) solution, or in a NaCl pH 5.5 solution. Under each condition, uptake in water-injected oocytes was subtracted from that in cRNA-injected oocytes to give SLC6A14-specific uptake (n= 20). ***P < 0.001 versus control.

Figure 3. Concentration-dependent β-alanine-induced current in SLC6A14-expressing oocytes

β-Alanine-induced current in two-electrode voltage-clamped oocytes was measured at −60 mV following exposure to β-alanine (0.2–20 mm, 2 min) in a NaCl pH 7.4 solution. Data are expressed as percentage current produced by 20 mmβ-alanine (n= 4).

Figure 4. Amino acid induced inward current in SLC6A14-expressing oocytes

A, example traces of amino acid-induced currents in SLC6A14-expressing and water-injected oocytes following exposure (2 min) to saturating concentrations (20 mm) of different amino acids in NaCl pH 7.4 solution. B, mean results for experiments described in A using SLC6A14-expressing oocytes where data are expressed as the percentage current induced by 20 mm leucine (n= 7).

Figure 5. Inhibition of SLC6A14-mediated β-alanine uptake by unlabelled amino acids

Inhibition of [³H]β-alanine (100 μm) transport into SLC6A14-expressing oocytes by various amino acids (all 2 mm) in NaCl pH 7.4 solution (n= 18–21). NS, P > 0.05; ***P < 0.001; all versus SLC6A14 control.

Figure 6. The effects of substrate N-methylation on β-alanine carrier-like transport by SLC6A14

SLC6A14-mediated [³H]β-alanine (100 μm) uptake (measured in NaCl pH 7.4 solution) in the presence of various amino acids (filled squares) and their N-methylated analogues (open squares) at 0.1, 1 and 10 mm. Amino acids are: A, leucine and N-methyl-leucine (N-Me-Leu); B, glycine and sarcosine (Sar; N-methyl-glycine); C, alanine and N-methyl-alanine (N-Me-Ala); D, AIB (aminoisobutyric acid) and MeAIB (α-(methylamino)isobutyric acid). Data are expressed as percentage of control (absence of competing amino acids) after subtraction of uptake in water-injected oocytes under each condition (n= 18–20).