Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers

Cytoplasmic Ca2+ is known to regulate Na+-Ca2+ exchanger (NCX) activity by binding to two adjacent Ca2+-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca2+-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca2+ decreases the distance between the cytoplasmic loops of NCX pairs. The Ca2+-dependent movements detected between paired NCXs were abolished by mutating the Ca2+ coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca2+ coordination site in CBD2 (E516L) had no effect. Thus, the Ca2+-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca2+-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca2+-dependent phenotype mirroring fulllength NCX but distinct from both CBD1 and CBD2.