Saturation Transfer Difference NMR experiments of membrane proteins in living cells under HR-MAS conditions: The interaction of the SGLT1 cotransporter with its ligands

In the last decade, Saturation Transfer Difference (STD)-NMR spectroscopy has been extensively exploited to study receptor-ligand interactions [1]. This technique has also been used to study molecular recognition events involving membrane receptors and their ligands, by working on samples which contained platelets or whole cells, and exploiting liquid state NMR [2,3]. This strategy allowed, at least in some particular cases, to overcome the inherent problems associated with the extreme difficulty of isolating and maintaining certain membrane receptors in solution with the correct folding and the proper functionality. Nevertheless, the authors employed platelets and cells, that live in suspension, particularly in the bloodstream, and that present a low tendency to aggregate and precipitate. In contrast, cells derived from solid tissues show a strong tendency to aggregate and precipitate which, in most cases, prevents the application of STD-NMR experiments. In fact, these kinds of cells remain in suspension for a very short time, leading to the acquisition of STD spectra of very poor quality. In this context, we aimed to develop a robust NMR methodology to study the interaction of ligands with membrane proteins, employing samples which contain whole and vital cells, as we considered important to have access to a method not affected by restrictions related to the nature of the tissue of origin. Therefore, the use of high resolution magic angle spinning (HR-MAS) NMR techniques has been explored to exploit the rotation at a relative high speed as a tool to maintain cells into the sample “active window”. Indeed, due to centrifuge force, cells do not sediment at the bottom of the NMR tube, as it occurs when a regular liquid state NMR probe is employed, but distribute along the internal walls of the MAS rotor, forming an homogeneous layer that mimics cell disposition in tissue surface [4]. To verify the feasibility of this approach, we selected a model system composed by the hSGLT1 cotransporter interacting with two of its known ligands. hSGLT1 is a Na+/glucose co-transporter membrane protein that uses the energy from a downhill sodium gradient to transport glucose across the apical membrane against an uphill glucose gradient. The glycoside phlorizin and naphthyl-β-d-galactoside are both competitive inhibitors of hSGLT1, but presenting affinities for the receptor that differ for more than three orders of magnitude [5]. In particular, tsA 201 cells were transfected with a vector containing a hSGLT1 encoding sequence (pEYFP-SGLT1). In order to obtain the cells for the STD control experiment, cells from the same line were transfected with the same plasmid, but containing no encoding insert, and thus merely expressing a basal level of hSGLT1. The phlorizin/hSGLT1 and naphthyl-β-d-galactoside/hSGLT1 interactions have been verified and characterized by STDD experiments acquired on sample containing the ligands and one of the cell lines described above [6]. Data obtained will be presented in this communication.