The second Ca ²⁺ -binding domain of the Na ⁺ –Ca ²⁺ exchanger is essential for regulation: Crystal structures and mutational analysis

Abstract

The Na ؉ -Ca 2؉ exchanger plays a central role in cardiac contractility by maintaining Ca 2؉ homeostasis. Two Ca 2؉ -binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca 2؉ binding to these regulatory domains activates the transport of Ca 2؉ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca 2؉ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca 2؉ -bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca 2؉ ions in primary and secondary Ca 2؉ sites. In the absence of Ca 2؉ , Lys 585 stabilizes the structure by coordinating two acidic residues (Asp 552 and Glu 648 ), one from each of the Ca 2؉ -binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca 2؉ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca 2؉ site completely remove Ca 2؉ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na ؉ -Ca 2؉ exchanger.calcium binding ͉ calcium regulation

Proton-sensing Ca2+ Binding Domains Regulate the Cardiac Na+/Ca2+ Exchanger

“…Structural studies clearly demonstrated that CBD domains do not undergo significant conformational changes upon Ca 2ϩ binding (7)(8)(9), although some specific (yet unidentified) electrostatic interactions of Ca 2ϩ with CBD sites may drive functionally important reorientation of two CBD domains (17,26,(33)(34)(35) sites of CBD1 are separated by 3-4 Å) and exhibit positive cooperativity for Ca 2ϩ binding despite the strong charge repulsion between the adjacent divalent cations (16,17,25,26,(33)(34)(35). The close adjacencies of Ca 2ϩ sites in CBDs (7)(8)(9)33) is consistent with a sharp dependence of Ca 2ϩ binding on pH (36 -38). The binding of the first Ca 2ϩ ion to CBD may partially (or fully) deprotonate the coordinating residue(s), thereby enabling the next Ca 2ϩ ion to bind to the remaining site(s).…”

“…Extreme Cooperativity of Ca 2ϩ -dependent Activation of I NCX in Ventricular Myocytes-A Hill coefficient of n H ϭ 2-3 has been reported for the Ca 2ϩ -dependent activation of I NCX in giant patches of cardiomyocytes and in oocyte expression systems (6,8,26,27). This cooperativity of the allosteric activation of NCX1 by Ca 2ϩ is consistent with the involvement of three Ca 2ϩ sites (among the six sites on the CBD12 tandem region) that regulate NCX1 (25,26 45 Ca binding to isolated CBDs (Figs.…”

“…The allosteric effect depends on Ca 2ϩ binding to a site that itself does not produce translocation but regulates transport kinetics. The cytosolic loop of NCX includes two closely spaced domains named Ca 2ϩ binding domain 1 (CBD1) and CBD2 (7)(8)(9), each of which share a common core structure typical of C 2 -type domains (10,11). Such C 2 domains are known to interact with diverse effectors (e.g.…”

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

“…The two high affinity (K d Ͻ 0.3 M) sites of CBD1 (Ca3 and Ca4) serve as the primary Ca 2ϩ sensor (7). A medium affinity site (CaI with a K d of 2-10 M) enables sustained NCX activation while also relieving Na ϩ -dependent inactivation (5,7,8). The site CaII of CBD2 serves as the Mg 2ϩ binding site (9,10), whereas the role of low affinity (K d Ͼ 10 M) sites of CBD1 (Ca1 and Ca2) remains unclear (7).…”

“…2). Further rise in cytosolic Ca 2ϩ may result in occupation of the CaI site at CBD2, alleviating Na ϩ -dependent inactivation and thus, allowing sustained NCX activation (5,8 …”