Structure and Dynamics of the Calcium Binding Domains of the Na/Ca Exchanger (NCX1.1) Determined by Site Directed Spin Labeling

The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion from cardiomyocytes following the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to the intracellular domain. This domain is composed of an α-catenin-like domain (CLD) that connects two structurally homologous Ca2+ binding domains (CBD1 and CBD2) to the transmembrane domain of the exchanger. NMR and X-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have suggested how Ca2+ binding alters their structures and motional dynamics. It remains unknown how Ca2+ binding to the intact Ca2+ sensor signals the transmembrane domain to regulate exchanger activity. We have used site directed spin labeling to address this question. Conventional EPR experiments have shown that: 1) residues in, or near, the Ca2+ binding loops of CBD1 and CBD2 show decreased mobility upon Ca2+ binding; and 2) residues in the β-sandwich regions are insensitive to Ca2+ binding. Double Electron Electron Resonance (DEER) measurements on doubly labeled constructs revealed that: 1) the structure of the β-sandwich domains of CBD1 and CBD2 are not altered upon Ca2+ binding; 2) CBD1 and CBD2 do not lie lengthwise antiparallel in close proximity but rather residues in the distal ends that connect to the CLD are greater than 60 A apart; and 3) residues nearer to the apex of the Ca2+ sensor are in close enough proximity to be measured by DEER and these distances are sensitive to Ca2+ binding. These studies support recent SAXS studies by Hilge et al. (PNAS 106:14333-8, 2009) and provide additional insight into a structural rearrangement of the intact Ca2+ sensor that may be involved in regulation of Na+/Ca2+ exchange.