The prokaryotic Na(+)/Ca(2+) exchanger NCX_Mj transports Na(+) and Ca(2+) in a 3:1 stoichiometry

Intracellular Ca(2+) signals control a wide array of cellular processes. These signals require spatial and temporal regulation of the intracellular Ca(2+) concentration, which is achieved in part by a class of ubiquitous membrane proteins known as sodium–calcium exchangers (NCXs). NCXs are secondary-active antiporters that power the translocation of Ca(2+) across the cell membrane by coupling it to the flux of Na(+) in the opposite direction, down an electrochemical gradient. Na(+) and Ca(2+) are translocated in separate steps of the antiport cycle, each of which is thought to entail a mechanism whereby ion-binding sites within the protein become alternately exposed to either side of the membrane. The prokaryotic exchanger NCX_Mj, the only member of this family with known structure, has been proposed to be a good functional and structural model of mammalian NCXs; yet our understanding of the functional properties of this protein remains incomplete. Here, we study purified NCX_Mj reconstituted into liposomes under well-controlled experimental conditions and demonstrate that this homologue indeed shares key functional features of the NCX family. Transport assays and reversal-potential measurements enable us to delineate the essential characteristics of this antiporter and establish that its ion-exchange stoichiometry is 3Na(+):1Ca(2+). Together with previous studies, this work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium–calcium exchangers.