Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides

Abstract

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na+. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl2, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The Km for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.

Inactivation by Hg2+ and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

“…Similar data were found in the presence of intraliposomal unconjugated and conjugated Hg 2+ and mersalyl (not shown). The data also correlates with the previous finding [27] that cysteine is transported from outside to inside (Fig. 7 A) but not vice versa (Fig.…”

“…7 A). This data indicated that cysteine was transported by the reconstituted ASCT2, as previously shown [27], whereas N-acetylcysteine and all the conjugates did not. In another experiment the uptake of [ 3 H]glutamine into proteoliposomes containing the same compounds externally added in the previous experiment was studied.…”

“…It was found that the reconstituted transporter is functionally and kinetically asymmetrical, is regulated by intraliposomal (intracellular) ATP and functions by a simultaneous mechanism of transport. In preliminary experiments it was also found that externally added mersalyl, p-hydroxymercurybenzoate and HgCl 2 inhibited the transporter [27][28]. Thus, the proteoliposome experimental model is suitable for investigating the effects of mercurial compounds on the ASCT2 transporter.…”

“…probably, cysteine SH group(s) of the reconstituted ASCT2 transporter are accessible from the external side of the proteoliposomes, which corresponds to the extracytoplasmic face of the transporter [27]. These functional groups could be involved in the mechanism of inhibition by mercurials.…”

“…It was reported that methylmercury modify the permeability to glutamine of cultured astrocytes by an indirect mechanism based on inhibition of expression of the amino acid transporters ASCT2 and SNAT3; however, this mechanism only partially explained the permeability alteration [26]. The rat glutamine/amino acid transporter ASCT2 which is expressed in kidney, intestine and nervous system besides other tissues [25], was reconstituted in liposomes [27][28]. The transporter was inserted unidirectionally in the proteoliposomal membrane, right-side-out with respect to the cell membrane; thus, the properties of the internal side of the reconstituted transporter correspond to those of the intracellular side and vice versa.…”