Received for publication November 14, 2006.
Revised February 1, 2007.
Accepted for publication February 1, 2007.

Conformationally Sensitive Reactivity to Permeant Sulfhydryl Reagents of Cysteine Residues Engineered into Helical Hairpin 1 of the Glutamate Transporter GLT-1

Abstract

In the central nervous system, glutamate transporters terminate the actions of this neurotransmitter by concentrating it into cells surrounding the synapse by a process involving sodium and proton cotransport followed by countertransport of potassium. These transporters contain two oppositely oriented helical hairpins 1 and 2. Hairpin 1 originates from the cytoplasm, but its tip is close to that of hairpin 2, which enters the transporter's lumen from the extracellular side. Here we address the question if hairpin 1 and/or domains surrounding it undergo conformational changes during the transport cycle. Therefore we probed the reactivity of cysteines introduced into hairpin 1 and the cytoplasmic ends of transmembrane domains 6, 7 and 8 of the GLT-1 transporter to membrane permeant N-ethylmaleimide. In each domain, except for transmembrane domain 6, cysteine mutants were found where the inhibition of D-[3H]-aspartate transport by the sulfhydryl reagent was increased when external sodium was replaced by potassium, a condition expected to increase the proportion of cytoplasmic-facing transporters. Conversely, the non-transportable blocker kainate protected against the inhibition in several of these mutants, presumably by locking the transporter in an outward-facing conformation. Moreover, external potassium decreased the oxidative crosslinking of two cysteines, each introduced at the tip of each hairpin. Our results are consistent with a model, based on the crystal structure of an archeal homologue. According to this model, the inward movement of hairpin 1 results in the opening a pathway between the binding pocket and the cytoplasm, lined by parts of transmembrane domains 7 and 8.

Key words: Amino Acid, Mutagenesis/Chimeric approaches

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