Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 2010-08-01

Passive water permeability of some wild type and mutagenized amino acid cotransporters of the SLC6/NSS family expressed in Xenopus laevis oocytes.

Massimo Santacroce, Michela Castagna, Vellea F Sacchi

Index: Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 156(4) , 509-17, (2010)

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Abstract

In this paper passive water movement across the cell membrane mediated by wild type and mutagenized cotransporters was investigated. We evaluated water movement and, in parallel, amino acid uptake induced by some members of the SLC6/NSS family belonging to different kingdoms, namely the rat GABA transporter GAT1, the insect amino acid transporters KAAT1 and CAATCH1 and the bacterial leucine transporter LeuT, whose structure was recently solved. We also tested whether mutated proteins in which the solute translocation mechanism is altered or even abolished were able to induce water movement across cell membrane. The proteins of interest were expressed in Xenopus laevis oocytes and osmotic water permeabilities were estimated from the rate of cell volume change induced by an osmotic gradient in the absence of cotransported solutes. Under osmotic stress all the studied wild type amino acid cotransporters increased the water permeability of the membrane. The GABA transport inhibitor SKF 89976A inhibited both GABA transport and water movement induced by the expression of GAT1. Interestingly, the capacity of mutant proteins to induce water movement was not predictable on the basis of their substrate transport ability. In particular the GAT1 mutant Q291N, void of any transport activity, induced a water permeability similar to that induced by the wt protein. The KAAT1 mutant T339C, which showed a higher transport activity, induced a water permeability not significantly different from the wild type transporter. Interestingly, the bacterial leucine cotransporter LeuT, whose binding site for leucine and Na(+) is void of water, induced water movement through the plasma membrane.Copyright (c) 2010 Elsevier Inc. All rights reserved.