Fig. 6. PATH has very different transport properties from CG1139 and mammalian PATs. (A,B) Histograms showing pH-dependent differences (P<0.001) in [³H]-alanine uptake by Xenopus laevis oocytes expressing either CG1139 (grey) or PATH (white) in acidic (pH 5.5) versus neutral (pH 7.4) medium, using 50 µM (A) and 75 nM (B) extracellular alanine (both well below the K_m of CG1139). (C,D) Histograms showing inhibition of [³H]-alanine uptake in CG1139-expressing (C; 50 µM [³H]-alanine, pH 5.5) and PATH-expressing (D; 75 nM [³H]-alanine, pH 7.4) oocytes by exogenous alanine, proline and glycine. All effects are statistically significant (P<0.03), except that of proline on PATH. (E) Self-inhibition of alanine uptake into oocytes expressing either CG1139 (50 µM [³H]-alanine, pH 5.5; black circles) or PATH (75 nM [³H]-alanine, pH 7.4; white circles). Inset shows data for PATH over low concentration range (0 to 0.25 mM). (F,G) Measurements of membrane potential from oocytes expressing CG1139 in the absence or presence of exogenous alanine, glycine and proline at pH 7.4 (F) and pH 5.5 (G). None of the other common amino acids produced a response. (H,I) Western blots carrying identical oocyte extracts probed with antibodies specific for activated S6K (H) and total S6K (I; phosphorylated and non-phosphorylated S6K are not fully separated in this gel). The blots were subsequently stained with Coomassie Blue to confirm equal loading. Oocytes incubated with external alanine (10 mM, 30 min; lane 1) produced no detectable signal, while S6K was activated by microinjection of 27 nl of 10 mM alanine (lane 2). Incubation of PATH-expressing oocytes in an alanine-containing solution (10 mM, 30 min; lane 3) also activated S6K.