Project 1: Molecular insights into the role of lipids and regulatory proteins on sodium coupling and inhibition of the GABA transporters BGT1 and GAT1

The project 1 concentrated on regulated transport of the osmolyte betaine in hyperosmotic stress response of renal cells. Medullar cells have to cope with extreme changes in extracellular osmolality from diuresis (low medullary solute concentrations) to antidiuresis (high medullary solute concentrations). The interstitial solution of the renal medulla is always fluctuant, and cells are constantly facing variable hypertonicity.

In general, cells counteract by changing the content of the remaining free cytoplasmic water through accumulation of ions and organic osmolytes facilitated by specific uptake systems, e.g. primary active ABC-transporters or secondary transporters. The equilibrium of both hydration and osmolyte concentration is an important physiological factor. Therefore, osmotically induced regulation of gene expression, metabolic activity and osmolyte transport is found in diverse tissues and organs, e.g. kidney, lung, cardiovascular system and musculoskeletal system, however the underlying mechanism and interplay of osmoregulated proteins is poorly understood.

Transporters of the neurotransmitter-sodium-symporter (NSS) or solute carriers 6 (SLC6) family facilitate osmolyte transport in mammals. The first SLC6 subfamily includes transporters for the inhibitory neurotransmitter GABA (h/m/rGAT1, SLC6A1; mGAT4 = h/rGAT3, SLC6A11; mGAT2 = h/rBGT1, SLC6A12 and mGAT3 = h/rGAT2, SLC6A13) and for the osmolytes betaine (mGAT2/BGT1, SLC6A12), taurine (mTauT, SLC6A6) and creatine (rCRET, SLC6A8). Among those only BGT1 is strongly osmo-regulated both on transcriptional and on transport level.

We aimed for an investigation of the regulatory roles of lipids and post-translational modifications on the osmolyte and GABA transport in BGT1. To do so we want to determine the structure of BGT1 and BGT1-mimicking BetP mutants to address the following questions in molecular detail:
Identification of a GABA, betaine and ion binding sites in BGT1 – We will determine the atomic structure of BGT1 by X-ray crystallography and cryo-EM single particle analysis. In addition, the bacterial betaine transporter BetP will serve as a structural homologue for BGT1 to explain its characteristics in sodium coupled osmolyte transport. We will use the BGT1-mimicking BetP mutants to investigate GABA coordination further and based on our homology model could predict a third sodium-binding site in BGT1, which might facilitate high betaine accumulation by tighter ion coupling in BGT1.

Regulatory role of BGT1 phosphorylation on serines and threonines - Protein kinase C (PKC) activation is known as regulator for many NSS transporters activating endocytosis-triggered depletion of transporters from the plasma membrane. To understand the influence of PKC phosphorylation on BGT1, several potential phosphorylation sites were mutated and tested with respect to their responses to PKC-mediated activators on the level of cellular distribution and transport properties. We will investigate these mutants structurally.

Regulatory role of N-glycosylation in BGT1 - N-glycosylation and its impact on the regulation mechanism of BGT1 was investigated in different cellular systems. We will continue studying the two predicted N-glycosylation sites (N171 and N183) in MDCK cells and oocytes and their individual role on BGT1 transport, regulation, stability and/or folding.