4064 C. M. H. Anderson and others J Physiol 586.17 earlier observations of the β-alanine carrier in rabbit ileum (K m = 2 mM) (Munck, 1985; Munck & Munck, 1992a). At saturating substrate concentrations (20 mM), β-alanine transport via SLC6A14 is associated with a large inward current of similar magnitude (P > 0.05) to that observed with the other dipolar substrate leucine (Fig. 4). The cationic amino acid lysine was associated with a larger current (P < 0.01, lysine versus β-alanine) which raises the possibility that a component of the current is carried by the positively charged amino acid (Fig. 4). MeAIB, which is excluded from the β-alanine carrier, failed to induce signiﬁcant current (P > 0.05) in the SLC6A14-injected oocytes (Fig. 4). None of the three β-alanine carrier substrates induced inward current in water-injected oocytes (Fig. 4A). The higher afﬁnity of SLC6A14 for lysine and leucine compared to β-alanine is conﬁrmed by the greater inhibition of [3H]β-alanine uptake observed (Fig. 5) when all competing cold substrates were present at a concentration (2 mM) around the K m for β-alanine (leucine and lysine both P < 0.001 versus cold β-alanine; β-alanine P < 0.001 versus control). A key functional characteristic of the β-alanine carrier in rabbit ileum (Munck, 1985) is the reduction in afﬁnity of the carrier for dipolar substrates following N -methylation. As measured in rabbit ileum (Munck, 1985), the effects of N -methylation on the ability of leucine, glycine, alanine and aminoisobutyric acid (AIB) to inhibit [3H]β-alanine transport via the β-alanine carrier were determined here in SLC6A14-injected oocytes (Fig. 6). An identical pattern of results was obtained in SLC6A14-injected oocytes (Fig. 6) to those measured 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. earlier for the β-alanine carrier in rabbit ileum (Munck, 1985). The ability of each amino acid to inhibit [3H]β-alanine transport was reduced by N -methylation. For example, greater inhibition of [3H]β-alanine uptake into SLC6A14-injected oocytes was observed at 10 mM for leucine (Leu) versus N -methyl-leucine (N -Me-Leu) (P < 0.001) (Fig. 6A), glycine (Gly) versus N -methyl-glycine or sarcosine (Sar) (P < 0.001) (Fig. 6B), alanine (Ala) versus N -methyl-alanine (N -Me-Ala) (P < 0.001) (Fig. 6C), and AIB versus MeAIB (P < 0.001) (Fig. 6D). Discussion The key functional characteristics of the β-alanine carrier, as previously described only in situ in rabbit ileum (Munck, 1985), are demonstrated following expression of the SLC6A14 transporter in Xenopus laevis oocytes (Figs 1–6). The transport system is both Na+ and Cl− dependent, can transport non-α-amino acids such as β-alanine with low afﬁnity, and has a higher afﬁnity for dipolar and cationic amino acids such as leucine and lysine (Figs 1–5). N -methylation of the substrates leucine, glycine, alanine and AIB reduces the afﬁnity for transport (Fig. 6). In addition, the characteristic of weak stereoselectivity has been demonstrated previously with the identiﬁcation of D-serine transport via SLC6A14 (Hatanaka et al. 2002). These observations conﬁrm the hypothesis that the SLC6A14 gene encodes the transport protein known as the β-alanine carrier (Munck, 1985; Anderson & Munck, 1987). The β-alanine carrier is considered to be the Na+-dependent transporter of leucine, lysine and alanine observed originally in rabbit ileum by Munck & Schultz (1969) and Paterson et al. (1981). Importantly, these observations also indicate that the studies of the β-alanine carrier in the 1960s to 1980s can now be considered to represent in situ measurements of SLC6A14 function in the intestine. The tissue distribution of the β-alanine carrier is unusual as the only intestinal location where function has been described is at the luminal surface of the ileum (Munck, 1985; Anderson & Munck, 1987; Munck & Munck, 1990, 1992a,b, 1995). This predominantly distal intestinal expression pattern is supported by observations using mouse gastrointestinal tract that demonstrate greater expression of SLC6A14 mRNA in the ileum and colon compared to duodenum and jejunum (Hatanaka et al. 2001, 2002; Ugawa et al. 2001; Sloan et al. 2003). In the rabbit, the physiological role of the β-alanine carrier seems certain to lie in nutrient absorption from the diet as the ileum is the site of maximal amino acid absorption in rabbit small intestine (Munck & Munck, 1992b). The physiological role in the human intestine is not known but the broad substrate speciﬁcity of SLC6A14 means that C 2008 The Authors. Journal compilation C 2008 The Physiological Society