The Na⁺-coupled System A amino acid transporters (SAT1-3) exhibit marked pH sensitivity, with influx depressed as external pH is lowered within the physiological range (Yao et al. 2000; Chaudhry et al. 2002); this may have important physiological implications for tissue amino acid and protein metabolism. (Bevington et al. 2002). We have investigated the possibility that this pH sensitivity relates to functionality of histidine residues within the transporter (SAT2) structure.

Rat SAT2 (Yao et al. 2000) was studied by overexpression in Xenopus laevis oocytes. SAT2 cRNA (50 ng) was injected into oocytes and experiments were performed 2-3 days later. L-[³H]Serine (0.5 mM) influx through SAT2 was measured at pH 7 and 8 with NaCl concentrations of 0-100 mM (TMACl added to maintain osmolarity). Values shown are means ± S.E.M.

Pre-treatment of SAT2-expressing oocytes with the histidine-modifying reagent diethylpyrocarbonate (DEPC) resulted in substantial reduction of serine transport activity (2 mM DEPC for 10 min produced maximal effect of 71 ± 5 % inhibition at pH 8; n = 8 expts) and loss of pH sensitivity over the pH range studied (Fig 1, right panel). DEPC treatment also blocks an increase in K_0.5 for Na⁺ activation of 0.5 mM serine transport seen on switching from pH 8 to 7 (Fig. 1, left panel). Na⁺ was not required for DEPC to inhibit transporter activity but the SAT2 substrate serine (5 mM) offered partial (~50 %) protection from the inactivating effects of DEPC. DEPC treatment abolishes pH sensitivity in the K_0.5 for Na⁺ activation of SAT2 without significantly affecting the value at pH 8. This is consistent with DEPC acting to alleviate an inhibitory allosteric effect of H⁺ on Na⁺ binding (Albers et al. 2001), rather than reduce competition between Na⁺ and H⁺ for a common binding site (Chaudhry et al. 2002). DEPC pre-treatment appears to reduce the rate of substrate translocation through SAT2, although the inability of DEPC to completely block transport suggests that its site of action is not directly at an essential substrate binding site. We suggest that DEPC binds to histidine residue(s) in the SAT2 protein at (or near) a putative H⁺ modifier site which may be in close proximity to the amino acid binding site (noting protection by serine).

We thank Dr J.D. Erickson (Louisiana State University) for rat SAT2 cDNA. This work was funded by MRC.