Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Baruch I. Kanner,Lihi Brocke,Annie Bendahan,Myriam Grunewald

doi:10.1074/jbc.m107735200

Baruch I. Kanner, Lihi Brocke

Open Access

https://doi.org/10.1074/jbc.m107735200

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Abstract

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.

Highlights

Important role in limiting the duration of synaptic excitation [7,8,9,10]
We report here the identification of two cysteine pairs, A412C/ V427C and A364C/S440C, which behave as if they are close in space
The Pairs A412C/V427C and A364C/S440C Create a High Affinity Binding Site for Cadmium (II) Ions—As a complementary approach to establish the proximity between cysteines introduced at positions 412 and 427 as well as between those at positions 364 and 440, we have examined the ability of the double mutants to form a high affinity Cd2ϩ binding site

Summary

Introduction

Important role in limiting the duration of synaptic excitation [7,8,9,10]. The uptake process is electrogenic [11,12,13], involving co-transport of three sodium ions, a proton, and a glutamate molecule and countertransport of a potassium ion (14 –16). Accessibility studies have shown that cysteine residues introduced at positions 364 and 440 of GLT-1, located on reentrant loops I and II, respectively, react with the impermeant sulfhydryl reagent MTSET added from the extracellular side (24 –26, 29). Pairs of cysteines were introduced in many combinations in the WT-GLT-1, and the double mutants were expressed in HeLa cells.

Results

Conclusion