Oligomerization of the GABA transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II*

The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the GABA-transporter-1 GAT1. We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer and β-lactamase fragment complementation. Of all generated mutants, only Y86A(but not Y86F-), E101A-, E101Qand E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Y86 and E101: Y86 participates in hydrophobic stacking. E101 is in the "a"-position of the leucine heptad repeat (where positions 1-7 are denoted "a-g" and each leucine is in the central "d" position). Thus, E101 is in the position predicted for the hydrogen bond donor, i.e. sandwiched between L97 and L104 which are one helical turn above and below E101. These key residues, namely Y86 and E101, are conserved in related transporters from archaeae to man; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments. 2 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from