Biochemical Characterization of Mammalian Sodium Calcium Exchangers

Ca2+ is a widespread second messenger in mammalian cells. It is crucial for various physiological processes. These include skeletal mineralization, cell proliferation, muscle cell contraction, nerve transmission, hormonal secretion, maintaining a regular heartbeat, and blood clotting. Na+/Ca2+ Exchangers (NCXs) play a central role in regulating the concentration of intracellular Ca2+ homeostasis through the high-capacity extrusion of Ca2+. In humans, three homologs of NCX exist, each has multiple cytosolic regulatory domains. The NCXs can also function in both monomeric and dimeric states. Dysfunction of NCXs has been associated with various diseases, including heart disease, neurodegenerative disorders, and cancer. This makes NCXs a highly promising therapeutic target. Despite extensive physiological research on this protein, the molecular principles governing the function and regulation of NCXs largely remain unknown due to limited structural studies. This effort is further hindered by the significant challenge of expressing and purifying both monomeric and dimeric NCXs. Using an improved mammalian expression system developed in our lab, we successfully expressed both monomeric and dimeric NCXs for all three homologs from both humans and rats, which were analyzed by fluorescence-detection size exclusion chromatography (FSEC). Additionally, we have expressed and purified 3C protease to facilitate the subsequent structural studies by removing the fluorescence and purification tags.