A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

Zhou Zhang,Thomas Albers,Heather L Fiumera,Armanda Gameiro,Christof Grewer

doi:10.1074/jbc.m109.038422

Abstract

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Highlights

Role in the brain [12], where it may help shuttle glutamine from astrocytes to neurons via the glutamate-glutamine cycle [1]
TM3 in particular and TM8 are long. This suggests a tilted configuration of these SNAT2 transmembrane helices (TMs) within the membrane bilayer, as found experimentally in LeuTAa and Mhp1 [21, 22]
Homology Modeling Suggests a Relationship with SLC5/6 Transporters—To further test the predicted distant relationship of SNAT2 and LeuTAa, we performed profile-based alignment using the HHSearch method, which has been shown to be efficient in finding distant relationships between protein families [26]

Summary

Introduction

Role in the brain [12], where it may help shuttle glutamine from astrocytes to neurons via the glutamate-glutamine cycle [1]. Homology Modeling Suggests a Relationship with SLC5/6 Transporters—To further test the predicted distant relationship of SNAT2 and LeuTAa, we performed profile-based alignment using the HHSearch method, which has been shown to be efficient in finding distant relationships between protein families [26].

Results

Conclusion