Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

“…To address these questions, we quantitatively evaluated the equilibrium conformations of the brain (CBD12-AD) and kidney (CBD12-BD) splice variants, as well as the E454K mutant (CBD12-AD-E454K) in the presence and absence of Ca 2ϩ by using EOM SAXS analysis and equilibrium 45 Ca 2ϩ binding assays. The only common sites among the constructs are the high affinity Ca3-Ca4 sites of CBD1 (8,14,18,25). Collectively, our data indicate that Ca 2ϩ binding to the Ca3-Ca4 sites results in domain rigidification through the population shift mechanism.…”

“…NCX proteins are expressed in a tissue-specific manner and contain an alternative splicing segment on CBD2, arising from a combination of six small exons, A, B, C, D, E, and F (1,11). In isolated CBD12 and intact NCX, CBD2 interacts with CBD1 (8,12,13), thereby modifying the equilibrium binding and kinetic properties of the primary allosteric sensor (Ca3-Ca4) in a splice-variant-dependent manner (14). Thus, Ca 2ϩ , the exchanger substrate, allosterically regulates the exchanger in a feedback manner.…”

“…The DNA constructs of CBD1-WT, CBD1-E454K, CBD12-AD, CBD12-AD-E454K, and CBD12-BD of canine NCX1 and CBD12-1.1 and CBD12-1.2 of CALX were expressed in Escherichia coli Rosetta2 (DE3) competent cells (Novagen), as described (10,14,18). Overexpressed proteins were purified on a Ni 2ϩ -nitrilotriacetic acid column followed by size exclusion chromatography on either Superdex-75 (CBD1 constructs) or Superdex-200 (CBD12 constructs) (Ͼ95% purity, judged by SDS-PAGE) (18).…”

“…The assay medium (1.5 ml) containing 7-15 M protein with 100 mM KCl, 10 mM Tris-HCl at pH 7.2 (TK buffer) was placed in the upper chamber of the Ultracel-3k concentrator, and the assay was performed as outlined previously (10,14 ] residual of decalcified buffer was measured by fluo-3 (10). All other procedures were described elsewhere (10).…”

“…Equilibrium 45 Ca 2ϩ Binding Assay-The equilibrium binding of 45 Ca 2ϩ to proteins was measured as the protein-bound radioactivity retained after ultrafiltration (10,14). The assay medium (1.5 ml) containing 7-15 M protein with 100 mM KCl, 10 mM Tris-HCl at pH 7.2 (TK buffer) was placed in the upper chamber of the Ultracel-3k concentrator, and the assay was performed as outlined previously (10,14 ] residual of decalcified buffer was measured by fluo-3 (10).…”