Abstract
Residues 386-423 of the rat brain serotonin transporter (SERT) are predicted to form a hydrophilic loop connecting transmembrane spans 7 and 8 (extracellular loop 4 or EL4). EL4 has been hypothesized to play a role in conformational changes associated with substrate translocation. To more fully investigate EL4 structure and function, we performed cysteine-scanning mutagenesis and methanethiosulfonate (MTS) accessibility studies on these 38 residues. Four EL4 mutants (M386C, R390C, G402C, and L405C) showed very low transport activities, low cell surface expression, and strong inhibition by MTS reagents, indicating high structural and functional importance. Twelve mutants were sensitive to very low MTS concentrations, indicating positions highly exposed to the aqueous environment. Eleven mutants were MTS-insensitive, indicating positions that were either buried in EL4 structure or functionally unimportant. The patterns of sensitivity to mutation and MTS reagents were used to produce a structural model of EL4. Positions 386-399 and 409-421 are proposed to form alpha-helices, connected by nine consecutive MTS-sensitive positions, within which four positions, 402-405, may form a turn or hinge. The presence of serotonin changed the MTS accessibility of cysteines at nine positions, while cocaine, a non-transportable blocker, did not affect accessibility. Serotonin-induced accessibility changes required both Na(+) and Cl(-), indicating that they were associated with active substrate translocation. With the exception of a single mutant, F407C, neither mutation to cysteine nor treatment with MTS reagents affected SERT affinities for serotonin or the cocaine analog beta-CIT. These studies support the role of EL4 in conformational changes occurring during translocation and show that it does not play a direct role in serotonin binding.
Highlights
neurotransmitter/sodium symporter (NSS) transporters couple the uptake of specific neurotransmitters and/or amino acids to the transmembrane gradients of Naϩ, ClϪ, and in some cases, Kϩ or Hϩ
Hypothetical model of EL4 —We have constructed a model for EL4 structure (Fig. 8) based on three major constraints: 1) positions 386 –399 and 409 – 421 were modeled as ␣-helices, based on the results of cysteine mutation, MTS sensitivity, and MTSEA biotinylation; 2) these ␣-helices were positioned so that residues 390 and 412, which form the Zn2ϩ binding site when replaced by histidine, were facing each other; and 3) these
␣-helices were placed next to each other so that their side chains were less than 4 Å apart, which is the average distance between binding coordinates in the Zn2ϩ binding sites of proteins with known three-dimensional structures [12]. (While the ability to form a Zn2ϩ binding site strongly limits the relative positions of these ␣-helices, it is possible that they could be significantly tilted with respect to each other, rather than lying parallel as depicted in the model.)
Summary
Materials—Plasmid DNA was prepared using Qiagen midiprep kits, and restriction fragments were purified from agarose gels using the QiaQuick gel extraction kit (Qiagen, Valencia, CA). The expression plasmid pRSTagM contains a promoter for T7 RNA polymerase upstream from the rat brain SERT cDNA This promoter was used to express wild-type and mutant SERTs in the vaccinia/T7 polymerase/HeLa cell system. Transport was measured by incubating the cells with 14.6 nM [1,2-3H(N)]serotonin (PerkinElmer Life Sciences) in 40 l of PBSCM for 20 min at room temperature. After 10 min of reaction at room temperature, the cells were washed three times with 100 l of PBSCM, assayed for transport or binding as described. When the ionic conditions were varied, stock solutions of serotonin and cocaine were diluted in buffer containing 8.6 mM K2HPO4, 2.8 mM KH2PO4, 0.1 mM CaCl2, 1 mM MgCl2, plus either 150 mM N-methyl-D-glucamine chloride (NMDG), 150 mM sodium isethionate or 150 mM NaCl. Control wells were subjected to the same ionic conditions in the absence of serotonin, cocaine, or MTS reagents. Bands were detected by chemiluminescence using Super Signal West Femto detection reagent (Pierce Chemical Co.)
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