Structural determinants of substrate specificity and Cl-role in two invertebrate cotransporters.

The two highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva of the lepidoptera Manduca sexta have been classified as members of the Na+/Cldependent transporter family (SLC6A gene family) (9; 11). These two cotransporters represent an interesting tool for investigating structurefunction relationships concerning ion and substrate selectivity in the SLC6 transporter family. At variance with the strict Na+ dependence of known vertebrate transporters, KAAT1 and CAATCH1 are able to exploit in addition a K+ electrochemical gradient to power up the uptake of neutral amino acids from the intestinal lumen. In fact, KAAT1 and CAATCH1 give rise to specific kinds of current depending on the transported amino acid, the cotransported ion, pH and the membrane voltage. Transportassociated currents with their own characteristics are induced by different organic substrates, which are notably distinct between the two proteins. Differences in amplitude, kinetics and voltagedependence of the transportassociated currents have been observed, as well as different substrate selectivity patterns, measured by radioactive amino acid uptake assays (9, 4, 23 ) In this work the specificity of KAAT1 was compared to that of CAATCH1, especially with respect to leucine, which in physiological conditions is transported by KAAT1, but not by CAATCH1, where it acts as a blocker. In order to identify the determinants involved in these phenomena, we have mutated the single different residue between the two transporters, the serine 308, that corresponds to that S290 participating in the leucine binding site of LeuTAa, a bacterial member of the SLC6A family of ioncoupled cotransporters (19). This amino acid is conserved only in the members of the family able to transport leucine ( 3), and its substitution in KAAT1 with threonine, blocks the current induced by leucine in sodium, and causes a reduction of about 70% of the current in potassium. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, that indicates the participation of this residue in the leucine binding site. The reverse mutation T308S in CAATCH1 conferred to this transporter the ability to transport leucine in presence of K+. The substrate specificity of KAAT1 has been investigated using electrophysiological and radiotracer methods. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine. These results may be interpreted by a kinetic scheme in which in presence of Na+ the leucinebound state of the transporter is relatively stable, while in presence of K+ and at negative potentials the progression of the leucinebound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inwardfacing conformation of the transporter, rather than in determining the specificity of leucine binding. Recent homology modeling work, based on the atomic structure of LeuTAa (19), has pinpointed the possible site of interaction of these proteins with chloride ions (25; 26 ). However, not all transporters in this family are Cldependent, and this difference appears to be due to the presence (or absence) of a critical negative charge in this region. This charge which might be provided either by the intrinsic glutamate side chain in the chlorideindependent transporters, or by a chloride ions in those transporters that have a nonconservative substitution in this position. The different chloride sensitivity observed in KAAT1 and CAATCH1, in spite of an identical sequence in the putative chloride binding site prompted us to re-examine this point. We have then performed electrophysiological experiments in which chloride was replaced either by iodide or by gluconate. Significant transportassociated current reductions were seen with gluconate replacement, while strong potentiation was induced by iodide. These observations indicate that for KAAT1 and CAATCH1 complete chloride dependency can be ruled out.