| Abstrakt: |
Until recently, insights into how mammalian secondary active ion-coupled solute transporters function at a molecular level have been limited. However, a number of prokaryotic transporter structures have been resolved and used to predict key functional characteristics in their mammalian counterparts [e.g. LeuT and the SLC6 family (Yamashita et al. 2005)]. The proton-coupled amino acid transporter PAT2 (slc36a2) is a member of the SLC36 family of transporters within the Amino Acid Auxin Permease superfamily (Thwaites & Anderson, 2011). PAT2 is expressed in the renal proximal tubule and defects in PAT2 contribute to iminoglycinuria (Bröer et al. 2008). To identify key functional elements within PAT2, an in silico homology modelling strategy was performed using crystal structures in the Protein Data Bank. HHPRED predicted a high degree of structural homology (>97%) for PAT2 and several prokaryotic transporter crystal structures, despite limited primary sequence identity (<20%), and differences in ion/substrate specificity and mode of action. Homology modelling of PAT2 against the LeuT template allowed prediction of functionally relevant amino acid residues. Site-directed mutagenesis of rat PAT2 was performed and transport function measured in Xenopus laevis oocytes. A key characteristic of secondary active transport is that during the transport cycle the substrate-bound pocket must be closed momentarily from both sides to prevent transmembrane leak. In LeuT, Y108 and F253 not only contribute to the substrate binding pocket but also form a molecular hatch that occludes bound substrate from the extracellular environment (Yamashita et al. 2005). In PAT2, homology modelling identified that Y163 and F272 occupy similar positions to those in LeuT. Mutation of either Y163 or F272 led to a decrease in PAT2 function. Concentration-dependent [3H]proline uptake (PAT2, Km 136 ± 27 µM, Vmax 329 ± 14 pmol/oocyte/40min, mean ± SEM N=3) showed reductions in both affinity and capacity for Y163F (Km 446 ± 115 µM, Vmax 93 ± 6 pmol/oocyte/40min) and F272Y (Km 253 ± 63 µM, Vmax 186 ± 11 pmol/oocyte/40min). Similar observations were made when function was measured as proline-induced inward current by TEVC. Confocal imaging revealed similar membrane localisation of FLAG-tagged PAT2, Y163F and F272Y suggesting that any change in activity was not due to decreased membrane expression. The conservation of tyrosine and phenylalanine residues in these two positions in many prokaryotic and eukaryotic transporters, combined with the changes in affinity (change in the binding pocket) and capacity (reduced ability to form an occluded bound-state), support the hypothesis that these residues play a gating role in PAT2, as observed previously in LeuT (Yamashita et al. 2005; Piscatelli et al. 2010). [ABSTRACT FROM AUTHOR] |
