Jonathan Lytton

Molecular Cloning of a Sixth Member of the K+-dependent Na+/Ca2+ Exchanger Gene Family, NCKX6

Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K+-dependent Na+/Ca2+ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the α-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K+-dependent Na+/Ca2+ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na+/Ca2+ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K+-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis in diverse tissues and cell types.

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in Motor Learning and Memory

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.

Differential contribution of plasmalemmal Na+/Ca2+ exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons

Inhibition of Na+,K+‐ATPase during NMDA applications greatly increased NMDA‐induced excitotoxicity in primary cultures of forebrain neurons (FNs), but not in cerebellar granule cells (CGCs). Because Na+,K+‐ATPase inhibition promotes reversal of plasmalemmal Na+/Ca2+ exchangers, we compared the activities of reversed K+‐independent (NCX) and K+‐dependent (NCKX) Na+/Ca2+ exchangers in these cultures. To this end, we measured gramicidin‐induced and Na+‐dependent elevation in cytosolic [Ca2+] ([Ca2+]c) that represents Ca2+ influx via reversed NCX and NCKX; NCX activity was dissected out by removing external K+. The [Ca2+]c elevations mediated by NCX alone, and NCX plus NCKX combined, were 17 and 6 times more rapid in FNs than in CGCs, respectively. Northern blot analysis showed that FNs preferentially express NCX1 whereas CGCs expressed NCX3. Differences in expression of other isoforms (NCX2, NCKX2, NCKX3 and NCKX4) were less pronounced. We tested whether the NCX or NCKX family of exchangers contributes most to the toxic NMDA‐induced Ca2+ influx in depolarized neurons. We found that in FNs, inhibition of NCX alone was sufficient to significantly limit NMDA excitotoxicity, whereas in CGCs, inhibition of both NCX and NCKX was required. The data suggest that the high activity of NCX isoforms expressed in FNs, possibly NCX1, sensitizes these neurons to NMDA excitotoxicity.

Stoichiometry of the cardiac Na+/Ca2+ exchanger NCX1.1 measured in transfected HEK cells

The stoichiometry with which the Na+/Ca2+ exchanger, NCX1, binds and transports Na+ and Ca2+ has dramatic consequences for ionic homeostasis and cellular function of heart mycocytes and brain neurons, where the exchanger is highly expressed. Previous studies have examined this question using native NCX1 in its endogenous environment. We describe here whole-cell voltage clamp studies using recombinant rat heart NCX1.1 expressed heterologously in HEK-293 cells. This system provides the advantages of a high level of NCX1 protein expression, very low background ion transport levels, and excellent control over clamped voltage and ionic composition. Using ionic conditions that allowed bi-directional currents, voltage ramps were employed to determine the reversal potential for NCX1.1-mediated currents. Analysis of the relation between reversal potential and external [Na+] or [Ca2+], under a variety of intracellular conditions, yielded coupling ratios for Na+ of 1.9-2.3 ions per net charge and for Ca2+ of 0.45 +/- 0.03 ions per net charge. These data are consistent with a stoichiometry for the NCX1.1 protein of 4 Na+ to 1 Ca2+ to 2 charges moved per transport cycle.

K+-dependent Na+/Ca2+ exchangers in the brain.

Abstract: Sodium‐calcium exchange was first characterized in heart myocytes and squid axon more than 3 decades ago. Since then, it has been appreciated that functioning of the Na/Ca exchanger molecule plays a critical role in calcium homeostasis in neurons. Genome analysis indicates that Na/Ca exchangers are a superfamily encoded by 7 different genes divided into 2 groups: the Na/Ca exchangers (NCX; SLC8) and the Na/Ca+K exchangers (NCKX; SLC24). Two different NCX genes, NCX1 and NCX2, are highly expressed in brain. We recently described the widespread expression of 2 NCKX‐type exchangers in brain, NCKX2 and NCKX3, and uncovered evidence for expression of another, NCKX4. The unique role that each different exchanger plays in neuronal calcium homeostasis, however, awaits further investigation. To begin exploring this central question, we examined both the expression pattern and the functional properties of the K‐dependent Na/Ca exchanger isoforms expressed in brain and compared and contrasted these with NCX‐type exchangers. Distinct patterns of transcript abundance, regional distribution, and developmental expression were noted for each isoform. Functional properties, including stoichiometry and the kinetic characteristics of ion binding, were determined for NCKX2 and are discussed in the context of cellular Ca2+ signaling.

A Circularized Sodium-Calcium Exchanger Exon 2 Transcript

Previous reports of Na/Ca exchanger gene 1 (NCX1) expression have revealed a major RNA transcript of 7 kilobase pairs (kb), minor transcripts of ∼13 and ∼4 kb, and a relatively abundant 1.8-kb RNA band. In the present report we demonstrate that the 1.8-kb message, which has a tissue and subcellular distribution matching that of full-length NCX1 but is not polyadenylated, corresponds to a perfectly circularized exon 2 species. The circular transcript contained the normal NCX1 start codon, a new stop codon introduced as a consequence of circularization, and encoded a protein corresponding to the NH2-terminal portion of NCX1, terminating just after amino acid 600 in the cytoplasmic loop. A linear version of the circular transcript was prepared and transfected into HEK-293 cells. A protein, matching the predicted size of ∼70 kDa, was expressed, and the transfected cells possessed Na/Ca exchange activity. Although in native tissue we could not detect a protein corresponding exactly to that predicted from the circular transcript, a prominent band of slightly shorter size, possibly representing further proteolytic processing of circular transcript protein, was observed in membranes from LLC-MK2 cells and rat kidney.

Alternative promoters and cardiac muscle cell-specific expression of the Na+/Ca2+exchanger gene

Many studies have investigated the regulation of the Na+/Ca2+exchanger, NCX1, but limited data exist on transcriptional regulation of the NCX1 gene. We have identified the transcription start sites of three tissue-specific alternative promoters of NCX1 transcripts from rat heart, kidney, and brain. We have characterized the cardiac NCX1 promoter, from which the most abundant quantities of NCX1 transcripts are expressed. Transfection of primary cardiac myocytes, CHO cells, and COS-7 cells with overlapping genomic DNA fragments spanning the NCX1 cardiac transcription start site has uncovered a cardiac cell-specific minimum promoter from -137 to +85. The cardiac NCX1 promoter is TATA-less but has putative binding sites for cardiac-specific GATA factors, an E box, and an Inr as well as multiple active enhancers. The kidney NCX1 promoter has a typical TATA box and binding sites for several tissue-specific factors. The brain NCX1 promoter is very GC-rich and possesses several Sp-1 binding sites consistent with its ubiquitous expression.Many studies have investigated the regulation of the Na+/ Ca2+ exchanger, NCX1, but limited data exist on transcriptional regulation of the NCX1 gene. We have identified the transcription start sites of three tissue-specific alternative promoters of NCX1 transcripts from rat heart, kidney, and brain. We have characterized the cardiac NCX1 promoter, from which the most abundant quantities of NCX1 transcripts are expressed. Transfection of primary cardiac myocytes, CHO cells, and COS-7 cells with overlapping genomic DNA fragments spanning the NCX1 cardiac transcription start site has uncovered a cardiac cell-specific minimum promoter from -137 to +85. The cardiac NCX1 promoter is TATA-less but has putative binding sites for cardiac-specific GATA factors, an E box, and an Inr as well as multiple active enhancers. The kidney NCX1 promoter has a typical TATA box and binding sites for several tissue-specific factors. The brain NCX1 promoter is very GC-rich and possesses several Sp-1 binding sites consistent with its ubiquitous expression.

Analysis of Ion Interactions with the K+ -dependent Na+/Ca+ Exchangers NCKX2, NCKX3, and NCKX4 IDENTIFICATION OF THR-551 AS A KEY RESIDUE IN DEFINING THE APPARENT K+ AFFINITY OF NCKX2

K+-dependent Na+/Ca2+ exchangers (NCKX) catalyze cytosolic Ca2+ extrusion and are particularly important for neuronal Ca2+ signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca2+ and Na+ were similar, NCKX3 and -4 displayed ∼40-fold higher affinities for K+ ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K+ affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca2+ affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K+ and Ca2+ affinities were reduced. These results indicate that the apparently low K+ affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K+ liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K+ binding pocket, because it was not possible to recreate the K+-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca2+ signaling.

Physiological and molecular characterization of the Na+/Ca2+exchanger in human platelets

In this report, we have demonstrated that Na+/Ca2+exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+exchanger could make platelets especially sensitive to daily fluctuations in salt intake.

Metabolic regulation of sodium–calcium exchange by intracellular acyl CoAs

The sodium–calcium exchanger (NCX) is a critical mediator of calcium homeostasis. In the heart, NCX1 predominantly operates in forward mode to extrude Ca2+; however, reverse‐mode NCX1 activity during ischemia/reperfusion (IR) contributes to Ca2+ loading and electrical and contractile dysfunction. IR injury has also been associated with altered fat metabolism and accumulation of long‐chain acyl CoA esters. Here, we show that acyl CoAs are novel, endogenous activators of reverse‐mode NCX1 activity, exhibiting chain length and saturation dependence, with longer chain saturated acyl moieties being the most effective NCX1 activators. These results implicate dietary fat composition as a plausible determinant of IR injury. We further show that acyl CoAs may interact directly with the XIP (exchanger inhibitory peptide) sequence, a known region of anionic lipid modulation, to dynamically regulate NCX1 activity and Ca2+ homeostasis. Additionally, our findings have broad implications for the coupling of Ca2+ homeostasis to fat metabolism in a variety of tissues.