We expressed full-length Na+-Ca2+ exchangers (NCXs) with mutations in two Ca2+-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca2+-dependent regulation of NCX. CBD1 has four ...Ca2+-binding sites, and mutation of residues Asp421 and Glu451, which primarily coordinate Ca2+ at sites 1 and 2, had little effect on regulation of NCX by Ca2+. In contrast, mutations at residues Glu385, Asp446, Asp447, and Asp500, which coordinate Ca2+ at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca2+. Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca2+ affinity but retained regulation, confirming a contribution of CBD2 to Ca2+ regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca2+ affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca2+-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca2+-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between β-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca2+, indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca2+-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca2+, although CBD2 is also necessary for Ca2+-dependent regulation.
Plasma membrane Na(+)-Ca2+ exchange is an essential component of Ca2+ signaling pathways in several tissues. Activity is especially high in the heart where the exchanger is an important regulator of ...contractility. An expanding exchanger superfamily includes three mammalian Na(+)-Ca2+ exchanger genes and a number of alternative splicing products. New information indicates that the exchanger protein has nine transmembrane segments. The exchanger, which transports Na+ and Ca2+, is also regulated by these substrates. Some molecular information is available on regulation by Na+ and Ca2+ and by PIP2 and phosphorylation. Altered expression of the exchanger in pathophysiological states may contribute to various cardiac phenotypes. Use of transgenic approaches is beginning to improve our knowledge of exchanger function.
The Na⁺-Ca²⁺ exchanger plays a central role in cardiac contractility by maintaining Ca²⁺ homeostasis. Two Ca²⁺-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity ...of the exchanger. Ca²⁺ binding to these regulatory domains activates the transport of Ca²⁺ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca²⁺ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca²⁺-bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca²⁺ ions in primary and secondary Ca²⁺ sites. In the absence of Ca²⁺, Lys⁵⁸⁵ stabilizes the structure by coordinating two acidic residues (Asp⁵⁵² and Glu⁶⁴⁸), one from each of the Ca²⁺-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca²⁺ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca²⁺ site completely remove Ca²⁺ 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⁺-Ca²⁺ exchanger.
The Na+/Ca2+ exchanger is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is governed by Ca2+, and the primary Ca2+ sensor (CBD1) is located ...in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. Here, we present a crystal structure of CBD1 at 2.5Å resolution, which reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing.
The superfamily of cation/Ca2+ exchangers includes both Na+/Ca2+ exchangers (NCXs) and Na+/Ca2+,K+ exchangers (NCKX) as the families characterized in most detail. These Ca2+ transporters have ...prominent physiological roles. For example, NCX and NCKX are important in regulation of cardiac contractility and visual processes, respectively. The superfamily also has a large number of members of the YrbG family expressed in prokaryotes. However, no members of this family have been functionally expressed, and their transport properties are unknown. We have expressed, purified, and characterized a member of the YrbG family, MaX1 from Methanosarcina acetivorans. MaX1 catalyzes Ca2+ uptake into membrane vesicles. The Ca2+ uptake requires intravesicular Na+ and is stimulated by an inside positive membrane potential. Despite very limited sequence similarity, MaX1 is a Na+/Ca2+ exchanger with kinetic properties similar to those of NCX. The availability of a prokaryotic Na+/Ca2+ exchanger should facilitate structural and mechanistic investigations.
Databases include many putative prokaryotic Na+/Ca2+ exchangers, but none have been functionally expressed.
A membrane protein (MaX1) of Methanosarcina acetivorans catalyzes electrogenic countertransport of Na+ and Ca2+.
MaX1 has properties similar to those of mammalian Na+/Ca2+ exchangers.
The characterization and purification of MaX1 should facilitate structure/function studies.
The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as the main mechanism for Ca2+ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca2+, NCX1.1 activity is also ...strongly regulated by Ca2+ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca2+ regulation. Nevertheless, the mechanisms by which Ca2+ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+. It was further demonstrated that a mutation of an acidic residue involved in Ca2+ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca2+ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca2+ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca2+ ions that bind to CBD1 are important for regulating exchanger activity.
Cardiac fibrillation, a form of cardiac arrhythmia, is the most common cause of embolic stroke and death associated with heart failure. The molecular mechanisms underlying cardiac fibrillation are ...largely unknown. Here we report a zebrafish model for cardiac fibrillation. The hearts of zebrafish tremblor (tre) mutants exhibit chaotic movements and fail to develop synchronized contractions. Calcium imaging showed that normal calcium transients are absent in tre cardiomyocytes, and molecular cloning of the tre mutation revealed that the tre locus encodes the zebrafish cardiac-specific sodium-calcium exchanger (NCX) 1, NCX1h. Forced expression of NCX1h or other calcium-handling molecules restored synchronized heartbeats in tre mutant embryos in a dosage-dependent manner, demonstrating the critical role of calcium homeostasis in maintaining embryonic cardiac function. By creating mosaic zebrafish embryos, we showed that sporadic NCX1h-null cells were not sufficient to disrupt normal cardiac function, but clustered wild-type cardiomyocytes contract in unison in tre mutant hearts. These data signify the essential role of calcium homeostasis and NCX1h in establishing rhythmic contraction in the embryonic zebrafish heart.
The cardiac Na+−Ca2+ exchanger (NCX1) is modeled to contain nine transmembrane segments (TMS) with a pair of oppositely oriented, conserved sequences called the α-repeats that are important in ion ...transport. Residue 122 in the α-1 repeat is in proximity to residue 768 in TMS 6, and the two residues can be cross-linked . During studies on the substrate specificity of this intramolecular cross-link, we found evidence that NCX1 can form dimers. At 37 °C in the absence of extracellular Na+, copper phenanthroline catalyzes disulfide bond formation between cysteines at position 122 in adjacent NCX1 proteins. Dimerization was confirmed by histidine tag pull-down experiments that demonstrate the association of untagged NCX1 with histidine-tagged NCX1. Dimerization occurs along a face of the protein that includes parts of the α-1 and α-2 repeats as well as parts of TMS 1 and TMS 2. We do not see cross-linking between residues in TMS 5, TMS 6, or TMS 7. These data provide the first evidence for dimer formation by the Na+−Ca2+ exchanger.
The Na(+)/Ca(2+) exchanger protein was first isolated from cardiac sarcolemma in 1988 and cloned in 1990. This allowed study of Na(+)/Ca(2+) exchange at the molecular level to begin. I will review ...the story leading to the cloning of NCX and the research that resulted from this event. This will include structure-function studies such as determination of the numbers of transmembrane segments and topological arrangement. Information on ion transport sites has been gathered from site-directed mutagenesis. The regions involved in Ca(2+) regulation have been identified, analyzed, and crystallized.We have also generated genetically altered mice to study the role of NCX in the myocardium. Of special interest are mice with atrial- or ventricular-specific KO of NCX that reveal new information on the role of NCX in excitation-contraction coupling and in cardiac pacemaker activity.
The Na(+)/Ca(2+) exchanger gene family encompasses three distinct proteins, NCX1, NCX2, and NCX3, which mediate cellular Ca(2+) efflux and thus contribute to intracellular Ca(2+) homeostasis. NCX1 is ...expressed ubiquitously while NCX2 and NCX3 are limited to brain and skeletal muscle. NCX1 exchanges 3 extracellular Na(+) for 1 intracellular Ca(2+). In addition to transporting Na(+) and Ca(2+), NCX1 activity is also regulated by these cations. NCX1 is especially important in regulating cardiac contractility.
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