Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K-0.5(Na+)) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K-0.5(Na+) at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.