Synaptic neurotransmission requires precise control of the release and reuptake of neurotransmitters. The reuptake is carried out by specific transporter proteins from the presynaptic plasma membrane. These transporters reuptake the neurotransmitters from the synaptic cleft, thereby inactivating both pre- and postsynaptic receptors. The transporters include the Na+/CI-dependent, serotonin transporter (SERT), dopamine transporter (DAT) and norepinephrine transporter (NET), all members of the neurotransmitter- sodium symporter (NSS) family. In addition to their key role in normal neurotransmission, these proteins interact directly with the widely prescribed antidepressants (e.g. fluoxetine (Prozac), imipramine and paroxetine) or psychostimulants (e.g. cocaine, amphetamine and MDMA (ecstasy)), all of them inhibit the neurotransmitter reuptake. In spite of their critical physiological, pharmacological and clinical importance, no high-resolution structural information is available for any NSS protein, partially due to the lacking of systems of overexpressing mammalian membrane proteins. To this end, we overexpressed and purified 5 out of 7 selected bacterial homologues of the mammalian neurotransmitter transporters in milligram quantities. We have identified one of them to be stable and monodisperse in a number of detergents and, thus, suitable for crystallization. The protein shares 20-25% sequence identity with the human transporters. We propose to grow two-dimensional crystals of this bacterial NSS protein and to determine its structure at approximately 10 A resolution using cryo-electron microscopy. This map should reveal the general architecture of the protein and its oligomeric state in the membrane. We also propose to crystallize the protein in three-dimensions to approximately 3 A resolution, and the crystals will be used to solve its high resolution structure using X-ray crystallography in the next stage of the project.
| Sauer, David B; Karpowich, Nathan K; Song, Jin Mei et al. (2015) Rapid Bioinformatic Identification of Thermostabilizing Mutations. Biophys J 109:1420-8 |
| Karpowich, Nathan K; Wang, Da-Neng (2013) Assembly and mechanism of a group II ECF transporter. Proc Natl Acad Sci U S A 110:2534-9 |
| Nyola, Ajeeta; Karpowich, Nathan K; Zhen, Juan et al. (2010) Substrate and drug binding sites in LeuT. Curr Opin Struct Biol 20:415-22 |
| Karpowich, Nathan K; Wang, Da-Neng (2010) Biophysics: Transporter in the spotlight. Nature 465:171-2 |
| Zhou, Zheng; Zhen, Juan; Karpowich, Nathan K et al. (2009) Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures. Nat Struct Mol Biol 16:652-7 |
| Karpowich, Nathan K; Wang, Da-Neng (2008) Structural biology. Symmetric transporters for asymmetric transport. Science 321:781-2 |
| Zhou, Zheng; Zhen, Juan; Karpowich, Nathan K et al. (2007) LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317:1390-3 |
