Struktur und Funktion des Betain Transporters BGT-1 aus der Niere
Final Report Abstract
In the course of this project we were able to investigate in detail the role of post-translational modifications on the regulatory insertion and depletion of BGT1. We could identify Thr40 and Asn183 as regulatory sites specific for BGT1-mediated stress response in kidney cells. Both work packages resulted in publications in recognized physiological journals, which were important to demonstrate our expertise on mammalian transporters in the field. We could establish the expression of BGT1 in Sf9 insect cells, but during our attempts to solve the BGT1 structure, structures of the eukaryotic dopamine and serotonin transporter were solved so we focussed on functional aspects and started to establish single-particle cryEM as structural biology method in our group. By working on BGT1 we managed to establish further mammalian protein expression. Our experience with BGT1 helped us to embark on other medical related projects, e.g., structure-function studies on a human TRP channel and on a human citrate transporter. Although we were not successful in obtaining a structure for BGT1 we were able to include homology modelling, which in the beginning of the project was still performed in collaboration with bioinformatics groups, to our toolbox. We are still working on a homology model-based structure-function study of BGT1 and the BGT1-mimicking BetP mutants. However, the mutagenesis studies were more challenging than anticipated. Most mutations studies failed because already wild type BGT1 yielded very small currents compared to its homologue GAT1, and mutating crucial transport hot spots like sodium binding sites diminished the currents below the detectable level. ITC and Fluorescence binding studies were not successful for betaine and sodium affinity studies in BGT1 either. Nonetheless we have now successfully established microscalethermophoresis (MST), which will help us to finish the still on-going last work packages – GABA and sodium coordination in BGT1. In addition we started single particle-cryoEM analysis on BGT1 in nanodiscs encouraged by the fact that we have successfully solved a new structure of BetP by this method this year. In summary the project “Structure and Function of BGT1” was of tremendously importance for my group and strengthened our standing in the field of mammalian transporters, which we could demonstrate by a well-received review article in 2012.
Publications
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Amino acid secondary transporters: toward a common transport mechanism. Curr Top Membr. 2012;70:1-28
Schweikhard ES, Ziegler CM
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(2013). Structural evidence for functional lipid interactions in the betaine transporter BetP. EMBO J. 32(23):3096-105
oshy C, Schweikhard ES, Gärtner RM, Perez C, Yildiz O, Ziegler C
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(2014). Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress. Am J Physiol Renal Physiol. 1;307(1):F107-15
Schweikhard ES, Kempson SA, Ziegler C, Burckhardt BC
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Role of N-glycosylation in renal betaine transport (2015). Biochem J. 470(2):169-79
Schweikhard ES, Burckhardt BC, Joos F, Fenollar-Ferrer C, Forrest LR, Kempson SA, Ziegler C
