Cytoplasmic segment interactions in the alpha(1)-subunit of the rat Na+, K+-ATPase

The currently accepted topographical model for the organization of the alph a -subunit of the Na+, K+-ATPase in the membrane considers that the protein has ten transmembrane segments and six cytoplasmic loops. Evidence of inte raction between the cytoplasmic regions may contribute to a better understa nding of the structure/function relationship of this protein. In this study , the first four cytoplasmic segments (C1, C2, C3 and C4) of the rat alpha 1 subunit were expressed in Escherichia Coli. The large cytoplasmic loop be tween transmembrane segments four and five (C3) retained its native structu re as demonstrated by the ability of ATP to protect against chemical modifi cation by Fluorescein 5-isothiocyanate (FITC). Interaction studies were con ducted by an overlay assay (Far Western blots) and surface plasmon resonanc e technology. We observed that C3 interacts with the N-terminal segment of the Na+, K+-ATPase, C1; and that both C1 and C3 interact with the cytoplasm ic segments C2 and C4.