A single residue controls the substrate-induced conductance in the glutamine transporter SNAT3 (slc38a3)

S. Bröer,¹ H.-P. Schneider,² A. Bröer¹ and J.W. Deitmer,² ¹School of Biochemistry & Molecular Biology, Australian National University, Building 41, Canberra, ACT 0200, Australia and ²Abteilung Allgemeine Zoologie, Fachbereich Biologie, Technical University Kaiserslautern, 67653 Kaiserslautern, Germany.

The glutamine transporter SNAT3 plays a pivotal role in several organs (Mackenzie & Erickson, 2004). In the liver it is involved in the uptake of glutamine, which serves as a central donor for ammonia dispatched in the urea cycle. In the brain SNAT3 releases glutamine from astrocytes and thereby forms part of the glutamate-glutamine cycle. In the kidney SNAT3 takes up glutamine into epithelial cells of the proximal tubule, where it is the principle donor of ammonia released in the urine to control plasma pH. The Mechanism of SNAT3 involves the cotransport of 1Na⁺ together with the substrate and the antiport of 1H⁺ (Broer et al., 2002). As a result the SNAT3 transporter could be regarded as a glutamine-dependent Na⁺/H⁺ antiporter. Although the overall mechanism of the transporter is electroneutral, substrate-induced currents are observed when the transporter is expressed in Xenopus laevis oocytes.* These ‘uncoupled’ currents are carried by cations or protons, depending on the extracellular pH (Schneider et al., 2007). To understand coupled and uncoupled movements of ions in the glutamine transporter SNAT3 we characterised site-directed mutants of the transporter. To identify residues that could be involved in Na⁺-binding and translocation we compared the sequences of proton-coupled transporters (solute carrier family [slc] 36) with those of Na⁺-coupled transporters (slc38). Residue Asn76 was selected for further analysis and changed into aspartate, glutamate, glutamine, histidine or serine. All of the mutants showed residual uptake of glutamine ranging from 10% to 40% of that of the wildtype. Subsequently, mutants were analysed in more detail by recording substrate-dependent currents, substrate-independent currents and by recording the intracellular pH using pH-sensitive microelectrodes. Mutation of Asn76 into any other residue completely abolished substrate-induced currents at pH 7.4, where they are largely carried by cations and at pH 8.4 where they are largely carried by protons. The glutamine-dependent Na⁺/H⁺ exchange activity by contrast was still readily detectable confirming the residual uptake of radioactive glutamine. The Asn76Gln replacement in particular showed significant coupled transport but no substrate-induced currents. Replacement of Asn76 by aspartate not only abolished substrate-induced currents but caused partial suppression of a substrate-independent conductance observed at pH 8.4. Removal of Na⁺ renders wildtype and mutant transporters non-functional, due to the Na⁺ cotransport mechanism. However, addition of glutamine in the absence of Na⁺ caused a huge increase of the membrane conductance in case of the Asn76Asp mutation but not in the wildtype. A model explaining these observations in terms of an alternate access mechanism of the SNAT3 protein has been developed.

Bröer A, Albers A, Setiawan I, Edwards RH, Chaudhry FA, Lang F, Wagner CA & Bröer S. (2002) Journal of Physiology, 539: 3-14.

Mackenzie B & Erickson JD. (2004) Pflügers Archiv (European Journal of Physiology), 447: 784-95.

Schneider HP, Bröer S, Bröer A & Deitmer JW. (2007) Journal of Biological Chemistry, 282: 3788-98.

*Procedures to remove oocytes were approved by the animal ethics committee of the ANU and the state of Rheinland-Pfalz.