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Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1

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Abstract

Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure–function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd2+) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + l-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

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Abbreviations

EAAT1:

Excitatory amino acid transporter 1

HP:

Hairpin loop

TM:

Transmembrane domain

CuPh:

Copper(II)(1,10-phenanthroline)3

TBOA:

d,l-Threo-β-benzyloxyaspartate

MTSET:

(2-Trimethylammonium)-methanethiosulfonate

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Acknowledgments

We thank Peng Zhang for making Fig. 6. This work was supported by the National Natural Science Foundation of China Grant 31170734, the International Centre for Genetic Engineering and Biotechnology Grant CRP/CHN11-01, the Specialized Research Fund for the Doctoral Program of Higher Education Grant 20114433120002, the Science and Technology Planning Project of Guangdong Province 2012B050200003, the Science and Technology Planning Project of Guangzhou 2013J4500018, and the Specialized Research Fund for talent introduction of colleges and universities of Guangdong Province Grant 2011430-1.

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The authors declare that they have no competing financial interests.

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Authors and Affiliations

  1. Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Yunlong Zhang & Shaogang Qu

  2. Teaching Center of Experimental Medicine, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Xiuping Zhang

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  1. Yunlong Zhang
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Correspondence to Shaogang Qu.

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Zhang, Y., Zhang, X. & Qu, S. Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1. Amino Acids 46, 1697–1705 (2014). https://doi.org/10.1007/s00726-014-1731-1

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