The H⁺-coupled amino acid transporter PAT1 has been cloned from rat (Sagné et al. 2001), mouse (Boll et al. 2002) and human (Chen et al. 2003). Recently, a related transporter (PAT2) was cloned from mouse and characterised functionally following expression in Xenopus laevis oocytes (Boll et al. 2002). Here we report the cloning and functional characterisation of rat PAT2.

A rat lung cDNA library was screened using a probe designed using the mouse PAT2 cDNA sequence (Boll et al. 2002). X. laevis were killed humanely and oocytes removed for expression studies. [³H]Amino acid (proline, alanine, glycine, MeAIB, leucine and lysine, all 100 µM, 5 µCi ml^-1) uptake (23 °C, 40 min, pH 5.0-7.4) was determined both in the presence and absence of extracellular Na⁺ in X. laevis oocytes 3 days after injection with 50 ng rat PAT2 cRNA.

A full-length 2396 bp cDNA (including polyA tail) was isolated from a rat lung cDNA library. The cDNA encodes for a transporter protein (rat PAT2), 481 amino acids in length. At the amino acid level rat PAT2 has 93 % identity with mouse PAT2 (Boll et al. 2002) and 75 % (over amino acids 52-477) with rat PAT1 (LYAAT-1) (Sagné et al. 2001). Proline uptake (pH 5.0-7.4) in rat PAT2 expressing X. laevis oocytes was greater than that in water-injected controls (P < 0.0001, ANOVA), uptake being pH dependent and significantly larger (P < 0.001) at pH 5.5 (71.67 ± 6.62 pmol oocyte^-1 (40 min)^-1 (20); mean ± S.E.M. (n)) than at pH 7.4 (35.25 ± 2.21 pmol oocyte^-1 (40 min)^-1 (19)). Uptake was Na⁺ independent at both pH 5.5 and 7.4 (P > 0.05, Na⁺ versus Na⁺-free conditions). Compared to PAT1, rat PAT2 is a high affinity transporter, K_m proline of 0.20 ± 0.04 mM. Measurement of uptake of a number of radiolabelled amino acids suggests that glycine, alanine and MeAIB are also substrates for rat PAT2 whereas leucine and lysine are not.

In conclusion, rat PAT2 was cloned from a rat lung cDNA library and functions as a high affinity pH-dependent, H⁺-coupled, Na⁺-independent amino acid transporter. The physiological role of this transporter requires further investigation.

This work was supported by the MRC (G9801704), BBSRC (13/D17277) and NIH (HL64196 & AI49849).