Identification of a chloride ion binding site in Na+/Cl -dependent transporters

Abstract

The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl(-) independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl(-) ions. However, the only Cl(-) ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK(A)s and homology modeling to predict the location of a functionally important Cl(-) binding site in serotonin transporter and other Cl(-)-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl(-) ion and through the observation of sequence conservation patterns in other Cl(-)-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl(-) and Na(+) ions during transport. Other implications of the model are also discussed.

Fig. 1.

Positions of negatively charged residues in aaLeuT. (A) Sequence of aaLeuT, with Asp and Glu residues in bold and underlined. TM segments (ends defined by using TMDET) and α-helices (defined by using DSSP) are indicated above the sequences. (B) View of aaLeuT from the plane of the membrane, with the extracellular side toward the top. The backbone of the protein is shown as a C^α trace, and side chains of Asp and Glu residues are shown by using sticks. Buried Glu residues are individually labeled. The approximate extent of the hydrophobic region of the membrane is indicated by brown lines.

Fig. 2.

Predicted protonation states of five buried Glu residues in aaLeuT. (A) E62–E419 pair. (B) E287–E290 pair. For the E62–E419 pair, the hydrogen is predicted to be midway between the carboxylate oxygen atoms of E62 or E419 and may be bonded to either atom. Potential hydrogen bonds are indicated by green dashed lines.

Fig. 3.

Predicted Cl⁻ binding site in eukaryotic Na⁺/Cl⁻-dependent transporters. (A) Model of Cl⁻ binding site in SERT. Distances (Å) are from oxygen or nitrogen atoms to the Cl⁻ ion (magenta sphere). Residues coordinating the Na1 site are also shown. (B) Model of the Cl⁻ binding site in aeAAT1. The Na⁺ ion in position Na1 is shown as a blue sphere. The backbone of four neighboring helices are shown as worms (TM 1, TM 2, and TM 6) or wire frame (TM 7).

Fig. 4.

Chloride dependence of SERT mutants. HeLa cells expressing wild-type SERT, S372A, S372C, and S372T (A) or S372D, S372E, and N368D (B) were assayed for 5-HT uptake for 10 min at the indicated Cl⁻ concentrations (as described in Materials and Methods). Transport activity is expressed relative to that at maximal Cl⁻ concentration. Each value represents the mean and SD of three independent experiments, each of which was performed in triplicate or quadruplicate wells.