The substrate specificity of KAAT1, a Na+- and K+-dependent neutral amino acid cotransporter cloned from the larva of the invertebrate Manduca sexta and belonging to the SLC6A gene family has been investigated using electrophysiological and radiotracer methods. The specificity of KAAT1 was compared to that of CAATCH1, a strictly related transporter with different amino acid selectivity. 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. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, constructed on the basis of the atomic structure of a leucine-transporting bacterial member of the family, which 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+. These results may be interpreted by a kinetic scheme in which, in presence of Na+, the leucine-bound state of the transporter is relatively stable, while in presence of K+ and at negative potentials the progression of the leucine-bound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity.
Structural and functional basis of amino acid specificity in the invertebrate cotransporter KAAT1 / A. Miszner, A. Peres, M. Castagna, S. Bettè, S. Giovannardi, F. Cherubino, E. Bossi. - In: THE JOURNAL OF PHYSIOLOGY. - ISSN 0022-3751. - 581:3(2007), pp. 899-913.
|Titolo:||Structural and functional basis of amino acid specificity in the invertebrate cotransporter KAAT1|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
|Data di pubblicazione:||2007|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1113/jphysiol.2007.132555|
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