Exploring PiTs: Functional and structural characterization of human SLC20 family members

• Applicant: Dr. Charlott Stock
• Subject Area: Structural Biology ; Biochemistry
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 462647097

The functional role and transport mechanism of sodium-dependent phosphate transporters of the SLC20 family are not well characterized. The two human SLC20 transporters PiT1 and PiT2 are omnipresent in the human body and their proposed functions range from regulators of Pi housekeeping to factors in adapted and genetic calcification disorders. Their importance is eminent because, a knockout of the PiT1 gene is embryonic lethal in mice and mutations of human PiT2 are linked to the severe genetic disease idiopathic basal ganglia calcification. In the absence of a production and purification strategy for human PiT1 and PiT2, functional studies are mostly limited to electrophysiological characterization of murine homologues in oocytes and only one structure from a bacterial homologue is available. The existing information on PiT transporters is not sufficient to understand their transport mechanism and does not allow any conclusion on the pathophysiological functions of disease-related mutants. I want to shed some light on the molecular details of PiT transporter function. I will implement a production and purification strategy for human PiT transporters, establish in vivo and vitro transport assays and obtain the first structures of these secondary active transporters from cryo-EM analysis. The four key aspects I will explore are the molecular details of the transport mechanism, the influence endogenous lipids have on transporter activity, the impact dimerization has on transporter function including possible cooperative effects and the function of the intracellular loop, which is only found in eukaryotic PiT transporters. My previous experience in membrane protein production, purification and handling in combination with the vast knowledge on secondary active transporters and excellent cryo-EM infrastructure present in the group of Prof. Poul Nissen and at Aarhus University make me confident that the proposed project is reasonable and feasible.

• DFG Programme: WBP Fellowship
• International Connection: Denmark
• Host: Professor Poul Nissen, Ph.D.