Debora A. Nicoll

Cloning of a Third Mammalian Na+-Ca2+ Exchanger, NCX3

NCX3 is the third isoform of a mammalian Na+-Ca2+ exchanger to be cloned. NCX3 was identified from rat brain cDNA by polymerase chain reaction (PCR) using degenerate primers derived from the sequences of two conserved regions of NCX1 and NCX2. The NCX3 PCR product was used to isolate two overlapping clones totalling 4.8 kilobases (kb) from a rat brain cDNA library. The overlapping clones were sequenced and joined at a unique Bsp106I restriction enzyme site to form a full-length cDNA clone. The NCX3 cDNA clone has an open reading frame of 2.8 kb encoding a protein of 927 amino acids. At the amino acid level, NCX3 shares 73% identity with NCX1 and 75% identity with NCX2 and is predicted to share the same membrane topology as NCX1 and NCX2. Following addition of a poly(A)+ tail to the NCX3 clone, exchanger activity could be expressed in Xenopus oocytes. NCX3 was also expressed in the mammalian BHK cell line. NCX3 transcripts are 6 kb in size and are highly restricted to brain and skeletal muscle. Linkage analysis in the mouse indicated that the NCX family of genes is dispersed, since the NCX1, NCX2, and NCX3 genes mapped to mouse chromosomes 17, 7, and 12, respectively.

A New Topological Model of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a “P-loop”-like structure.

Sodium-calcium exchange.

During the past year, significant advances have been made in the investigation of molecular, kinetic and electrophysiological aspects of Na(+)-Ca2+ exchange. The cardiac and retinal exchangers have been cloned and structure-function studies have begun.

Functional Adult Myocardium in the Absence of Na+-Ca2+ Exchange. Cardiac-Specific Knockout of NCX1

The excitation–contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

Mutation of Amino Acid Residues in the Putative Transmembrane Segments of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

We have examined the role of conserved regions and acidic or basic residues located in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger by site-directed mutagenesis. The α-1 and α-2 repeats are transmembrane regions of internal similarity, which are highly conserved among Na+-Ca2+ exchangers. We find that Na+-Ca2+ exchange activity is highly sensitive to mutagenesis in the α-repeats. Mutation at residues Ser-109, Ser-110, Glu-113, Ser-139, Asn-143, Thr-810, Ser-811, Asp-814, Ser-818, or Ser-838 resulted in loss of exchanger activity. Mutation at residues Thr-103, Gly-108, Pro-112, Glu-120, Gly-138, Gly-809, Gly-837, and Asn-842 resulted in reduced exchanger activity, and altered current-voltage relationships were observed with mutations at residues Gly-138 and Gly-837. Only mutation at residue Ser-117 appeared to leave exchanger activity unaffected. Thus, the α-repeats appear to be important components for ion binding and translocation. Another region implicated in exchanger function is a region of similarity to the Na+,K+ pump (Nicoll, D. A., Longoni, S., Philipson, K. D. (1990) Science 250, 562-565). Mutations at two residues in the pump-like region, Glu-199 and Thr-203, resulted in nonfunctional exchangers, while mutation at two other residues, Glu-196 and Gly-200, had no effect. The role of acidic and basic residues in the transmembrane segments was also examined. Mutation of several basic residues (Arg-42, His-744, Lys-751, Lys-797, and His-858) did not affect exchange activity. Of the acidic residues located outside of the α-repeat and pump-like regions (Asp-740, Asp-785, and Asp-798), only mutation at Asp-785 resulted in reduction of exchanger activity.

The Na+/Ca2+ Exchange Molecule

Abstract: An overview of the molecular physiology of the Na+/Ca2+ exchanger is presented. This includes information on the variety of exchangers that have been described and their regulatory properties. Molecular insight is most detailed for the cardiac Na+/Ca2+ exchanger (NCX1). Parts of the NCS1 molecule involved in regulation and ion transport have been elucidated, and initial information on the topology and structure is available.

The sodium/calcium exchanger family—SLC8

The Na+/Ca2+ exchanger gene family encompasses three distinct proteins, NCX1, NCX2, and NCX3, which mediate cellular Ca2+ efflux and thus contribute to intracellular Ca2+ homeostasis. NCX1 is expressed ubiquitously while NCX2 and NCX3 are limited to brain and skeletal muscle. NCX1 exchanges 3 extracellular Na+ for 1 intracellular Ca2+. In addition to transporting Na+ and Ca2+, NCX1 activity is also regulated by these cations. NCX1 is especially important in regulating cardiac contractility.

The Crystal Structure of the Primary Ca2+ Sensor of the Na+/Ca2+ Exchanger Reveals a Novel Ca2+ Binding Motif

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 second Ca2+-binding domain of the Na+–Ca2+ exchanger is essential for regulation: Crystal structures and mutational analysis

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.

Functional Analysis of a Disulfide Bond in the Cardiac Na+-Ca2+ Exchanger

The electrophoretic mobility of the cardiac Na+-Ca2+ exchange protein is different under reducing and nonreducing conditions. This mobility shift is eliminated in a cysteine-less exchanger, suggesting that the presence or absence of an intramolecular disulfide bond alters the conformation and mobility of the exchanger. Using cysteine mutagenesis and biochemical analysis, we have identified the cysteine residues involved in the disulfide bond. Cysteine 792 in loop h of the exchanger forms a disulfide bond with either cysteine 14 or 20 near the NH2terminus. Because the NH2 terminus is extracellular, the data establish that loop h must also be extracellular. A rearrangement of disulfide bonds has previously been implicated in the stimulation of exchange activity by combinations of reducing and oxidizing agents. We have investigated the role of cysteines in the stimulation of the exchanger by the combination of FeSO4 and dithiothreitol (Fe-DTT). Using the giant excised patch technique, we find that stimulation of the wild type exchanger by Fe-DTT is primarily due to the removal of a Na+-dependent inactivation process. Analysis of mutated exchangers, however, indicates that cysteines are not responsible for stimulation of the exchange activity by Fe-DTT. Ca2+ blocks modification of the exchanger by Fe-DTT. Disulfide bonds are not involved in redox stimulation of the exchanger, and the modification reaction is unknown. Modulation of Na+-dependent inactivation may be a general mechanism for regulation of Na+-Ca2+ exchange activity and may have physiological significance.