4062 C. M. H. Anderson and others J Physiol 586.17 system B0,+ (identiﬁed originally in mouse blastocysts; Van Winkle et al. 1985). Thus far, β-alanine carrier-like function has not been demonstrated by any solute carrier transport system identiﬁed at the molecular level. In this investigation, a series of experiments were designed to determine whether SLC6A14 is the molecular correlate of the intestinal β-alanine carrier, perhaps the last of the classical intestinal amino acid transport systems to be identiﬁed at the molecular level. Methods Materials [3H]β-Alanine (50 Ci mmol−1) was from American Radiolabelled Chemicals. [3H]Lysine (99 Ci mmol−1), [3H]leucine (115 Ci mmol−1) and [14C]MeAIB (α-(methylamino)isobutyric acid) (51 mCi mmol−1) were from PerkinElmer. Functional expression in Xenopus laevis oocytes Human SLC6A14 cRNA was produced by in vitro transcription (using mMessage mMachine T7 Ultra kit (Ambion)) of pSPORT1 plasmid containing the SLC6A14 sequence isolated originally from MCF-7 cells (Nakanishi et al. 2001). Female Xenopus laevis were killed humanely by cervical dislocation following Schedule 1 procedures. Oocytes were prepared and injected with 50 nl cRNA (1 mg ml−1) or water, as previously described (Kennedy et al. 2002, 2005), and incubated at 18◦C in Barth’s solution until required. 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. Uptake of radiolabelled amino acids Uptake of radiolabelled amino acids (2–5 μCi ml−1) was measured in oocytes 2–5 days after injection, as previously described (Kennedy et al. 2002). Oocytes were washed in a NaCl-containing pH 7.4 solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes adjusted to pH 7.4 with Tris base) and uptake measured at 22◦C for 40 min. Uptake measurements were performed in this NaCl-containing pH 7.4 solution or using a solution adjusted as follows: for the Na+-free solution, NaCl was replaced with choline chloride; for the Cl−-free solution, NaCl, KCl, CaCl2 and MgCl2 were replaced with sodium gluconate, potassium gluconate, calcium gluconate and MgSO4, respectively; for the pH 5.5 solution, Hepes was replaced by Mes. After uptake, oocytes were washed three times in ice-cold buffer and lysed in 10% SDS. Radio- activity was measured by scintillation counting. Two-electrode voltage clamp Oocytes (2–8 days post-injection) were superfused in an open chamber with a NaCl-containing pH 7.4 solution (see above). Oocytes were clamped at −60 mV and exposed to various concentrations of β-alanine (0.2–20 mM, 2min) or different amino acids (all at 20 mM, 2min) to allow amino acid-induced currents to be measured using a Geneclamp 500 ampliﬁer, Digidata 1200 (Axon Instruments) and Clampex software (Kennedy et al. 2005). Currents were analysed using Clampﬁt 8.2. To determine the current evoked by a 2 min exposure to an amino acid, the current measured over the last 15 s of the 2 min exposure was averaged. The baseline current (taken as the average current over the 15 s before exposure to the amino acid) was then subtracted to determine SLC6A14-speciﬁc current. Statistics Data are means ± S.E.M. Statistical comparisons were made using ANOVA and Tukey’s post hoc test using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA). Curves were ﬁtted with GraphPad Prism 4. Results At a tracer concentration of 2 μM there was a 9.5-fold increase in [3H]β-alanine uptake in SLC6A14 cRNA-injected oocytes compared to water-injected oocytes (P < 0.01) (Fig. 1). Similarly, other amino acids identiﬁed as substrates for the β-alanine carrier in rabbit ileum (Munck, 1985; Munck & Munck, 1992b) also showed signiﬁcant uptake into SLC6A14-injected oocytes with 5.7-fold and 4.1-fold increases in uptake, compared C 2008 The Authors. Journal compilation C 2008 The Physiological Society