Modulation of the substrate selectivity of ASCT1 at low pH

Full text: The alanine,serine, cysteine transporter 1 (ASCT1) and the glutamate transporters belong to a family of structurally related membrane proteins. ASCT1 selectively transports small neutral amino acids, whilst the glutamate transporters are selective for acidic amino acids. A unique feature of ASCT1 is that at low pH (<7.0), anionic amino acids such as cysteate, can inhibit the uptake of neutral amino acids. This study investigated whether cysteate is a substrate or a non-transportable blocker of ASCT1 at low pH. Recombinant ASCT1 was expressed in oocytes from Xenopus leavis frogs anaesthetised with 0.17% 3-aminobenzoic acid ethyl ester. Transporter function was assayed 3-6 days later by two-electrode voltage clamp recording and radiolabelled flux experiments. Although substrate transport by ASCT1 is electroneutral, the transport process also activates an uncoupled anion conductance. In voltage clamped oocytes L-alanine activates a current that is maximal at pH 7.0 and decreases with decreasing pH, while L-cysteate activates a current which is not detected at pH 7.5 and reaches a maximum at pH 5.0. Oocytes expressing ASCT1 were injected with 3H- alanine and exchange was initiated by the application of external unlabelled substrate. At pH 7.5 external L-alanine initiates 3H-alanine release,while external L-cysteate has minimal effect on release. In contrast, at pH 6.0 external L-cysteate causes significant release of 3H-alanine above background. These results demonstrate that the substrate selectivity of ASCT1 is pH dependent, and raise the question of how substrate selectivity is achieved by these transporters and also that of the closely related glutamate transporters. Copyright (2001) Australian Neuroscience Society