Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC181

Poster Communications

Identification of tryptophan analogues as non-transported inhibitors of the proton-coupled amino acid transporter PAT2 (slc36a2)

N. Edwards1, C. Anderson1, D. T. Thwaites1

1. Epithelial Research Group, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom.

PAT2 (slc36a2) is a pH-dependent, Na+-independent transporter of small, dipolar amino and imino acids (1,2). PAT2 is defective in the renal reabsorptive disorder iminoglycinuria (3). Tryptophan analogues act as non-transported inhibitors of the amino acid transporters PAT1 (4) and ATB0,+ (5). The purpose of this study was to identify the nature of the interaction of tryptophan analogues with PAT2. Rat PAT2 was expressed in X. laevis oocytes following standard procedures (2). Data are mean ± SEM (n) with statistical significance determined by ANOVA and Tukey’s multiple comparison post-test. Under optimal uptake conditions (pH 5.5, Na+-free), 5-hydroxy-L-tryptophan and α-methyl-DL-tryptophan both significantly reduce (p<0.001) [3H]proline uptake (10μM, 5μ, 40min, 22°C) in PAT2-expressing oocytes in a concentration-dependent manner with IC50 values of 4.2 ± 0.8 (20) and 3.5 ± 0.6 (30) mM, respectively. Rheogenic H+/proline symport via PAT2 was measured using two-electrode voltage-clamp (-60mV, pH 5.5, Na+-free). In contrast, 5-hydroxy-L-tryptophan and α-methyl-DL-tryptophan (both 20mM) failed to induce inward current in PAT2-expressing oocytes. However, both 5-hydroxy-L-tryptophan and α-methyl-DL-tryptophan (20mM) significantly reduced (p<0.001) the proline- (0.2mM) induced current by 81.0 ± 2.3 (6) and 76.7 ± 3.6 (5) %, respectively. To confirm that both tryptophan analogues are non-transported inhibitors of PAT2, trans-stimulation of [3H]proline efflux (oocytes were preloaded with 5mM proline, 0.1μ was measured under optimal uptake conditions. The PAT2 substrate glycine (10mM) significantly trans-stimulated (p<0.001) [3H]proline efflux via PAT2 [266.5 ± 17.2 (10) pmol.oocyte-1.(10min)-1] compared to water-injected oocytes [13.6 ± 2.5 (10) pmol.oocyte-1.(10min)-1]. In contrast, both 5-hydroxy-L-tryptophan and α-methyl-DL-tryptophan (both 10mM) failed (p>0.05 versus water) to trans-stimulate [3H]proline efflux via PAT2 [9.3 ± 1.0 (10) and 11.9 ± 1.4 (10) pmol.oocyte-1.(10min)-1, respectively]. In conclusion, the combination of measurements demonstrate that 5-hydroxy-L-tryptophan and α-methyl-DL-tryptophan are non-transported inhibitors of PAT2. These compounds should prove useful experimental tools to help elucidate the physiological role of PAT2.

Where applicable, experiments conform with Society ethical requirements