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J. Biol. Chem., Vol. 280, Issue 38, 32649-32654, September 23, 2005

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Substituted Cysteine Accessibility of the Third Transmembrane Domain of the Creatine Transporter

DEFINING A TRANSPORT PATHWAY^*

From the Cell Biology and Biochemistry Section, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na⁺ and Cl^- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an -helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321–28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na⁺- and Cl^--dependent neurotransmitter transporters.

Received for publication, June 21, 2005 , and in revised form, July 14, 2005.

^* This work was supported by grants from the Auckland Medical Research Foundation, Health Research Council of New Zealand, and Royal Society of New Zealand Marsden Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 64-9-3737599 (ext. 88009); Fax: 64-9-3737414; E-mail: d.christie{at}auckland.ac.nz.

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