The Na+/Ca2+exchanger in Alzheimer’s disease

As a pivotal player in regulating sodium (Na⁺) and calcium (Ca²⁺) homeostasis and signalling in excitable cells, the Na⁺/Ca²⁺ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca²⁺ and/or Na⁺ concentrations occurs, including Alzheimer’s disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca²⁺ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na⁺ elevation occurring in several pathophysiological conditions. Since the alteration of Na⁺ and Ca²⁺ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aβ), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aβ insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy.     

Cellular and subcellular localization of Na+-Ca2+ exchanger protein isoforms, NCX1, NCX2, and NCX3 in cerebral cortex and hippocampus of adult rat

Na(+)-Ca(2+) exchanger (NCX) controls cytosolic Ca(2+) and Na(+) concentrations ([Ca(2+)](i) and [Na(+)](i)) in eukaryotic cells. Here we investigated by immunocytochemistry the cellular and subcellular localization of the three known NCX isoforms, NCX1, NCX2 and NCX3, in adult rat neocortex and hippocampus. NCX1-3 were widely expressed in both brain areas: NCX1 immunoreactivity (ir) was exclusively associated to neuropilar puncta, while NCX2-3 were also detected in neuronal somata and dendrites. NCX1-3 ir was often identified around blood vessels. In both neocortex and hippocampus, all NCX isoforms were prominently expressed in dendrites and dendritic spines contacted by asymmetric axon terminals, whereas they were poorly expressed in presynaptic boutons. In addition, NCX1-3 ir was detected in astrocytes, notably in distal processes ensheathing excitatory synapses. All NCXs were expressed in perivascular astrocytic endfeet and endothelial cells. The robust expression of NCX1-3 in heterogeneous cell types in the brain in situ emphasizes their role in handling Ca(2+) and Na(+) in both excitable and non-excitable cells. Perisynaptic localization of NCX1-3 in dendrites and spines indicates that all isoforms are favourably located for buffering [Ca(2+)](i) in excitatory postsynaptic sites. NCX1-3 expressed in perisynaptic glial processes may participate in shaping astrocytic [Ca(2+)](i) transients evoked by ongoing synaptic activity.     

Silencing or knocking out the Na(+)/Ca(2+) exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.     

BHK cells transfected with NCX3 are more resistant to hypoxia followed by reoxygenation than those transfected with NCX1 and NCX2: Possible relationship with mitochondrial membrane potential

The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.     

Expression of Na(+)/Ca(2+) exchanger isoforms (NCX1 and NCX3) and plasma membrane Ca(2+) ATPase during osteoblast differentiation

The ability to deliver calcium to the osteoid is critical to osteoblast function as a regulator of bone calcification. There are two known transmembrane proteins capable of translocating calcium out of the osteoblast, the Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). In this study, we reveal the presence of the NCX3 isoform in primary osteoblasts and examine the expression of NCX1, NCX3, and PMCA1 during osteoblast differentiation. The predominant NCX isoform expressed by osteoblasts is NCX3. NCX1 also is expressed, but at low levels. Both NCX isoforms are expressed at nearly static levels throughout differentiation. In contrast, PMCA expression peaks at 8 days of culture, early in osteoblast differentiation, but declines thereafter. Immunocytochemical co-detection of NCX and PMCA reveal that NCX is positioned along surfaces of the osteoblast adjacent to osteoid, while PMCA is localized to plasma membrane sites distal to the osteoid. The expression pattern and spatial distribution of NCX support a role as a regulator of calcium efflux from osteoblasts required for calcification. The expression pattern and spatial distribution of PMCA makes its role in the mineralization process unlikely and suggests a role in calcium homeostasis following signaling events.     

Expression of the sodium/calcium exchanger in mammalian skeletal muscle cells in primary culture

Previous investigations have demonstrated molecular and functional expression, at early phases of development of skeletal muscle cells in primary culture, of cardiac isoforms of proteins involved in calcium transport and regulation, like the L-type calcium channel. Here the expression of the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1) was studied in skeletal muscle cells developing in vitro, by using biochemical, immunological, and electrophysiological techniques. Northern and Western blot experiments revealed the presence of this cardiac exchanger and its increasing expression during the early phases of development. Confocal imaging of myotubes showed an NCX1 distribution that was predominantly sarcolemmal. The whole-cell patch-clamp technique allowed us to record ionic currents, the direction and the amplitude of which depended on extracellular sodium and calcium concentrations. The developmental changes of this functional expression could be correlated with the molecular NCX1 expression changes. Taken together these data demonstrate the presence of the NCX1 isoform of the Na(+)/Ca(2+) exchanger during in vitro myogenesis and reinforce the theory that significant levels of cardiac-type proteins are transiently expressed during the early phases of the skeletal muscle cell development.     

Genetically modified mice to unravel physiological and pathophysiological roles played by NCX isoforms

Since the discovery of the three isoforms of the Na⁺/Ca²⁺ exchanger, NCX1, NCX2 and NCX3 in 1990s, many studies have been devoted to identifying their specific roles in different tissues under several physiological or pathophysiological conditions. In particular, several seminal experimental works laid the foundation for better understanding gene and protein structures, tissue distribution, and regulatory functions of each antiporter isoform. On the other hand, despite the efforts in the development of specific compounds selectively targeting NCX1, NCX2 or NCX3 to test their physiological or pathophysiological roles, several drawbacks hampered the achievement of these goals. In fact, at present no isoform-specific compounds have been yet identified. Moreover, these compounds, despite their potency, possess some nonspecific actions against other ion antiporters, ion channels, and channel receptors. As a result, it is difficult to discriminate direct effects of inhibition/activation of NCX isoforms from the inhibitory or stimulatory effects exerted on other antiporters, channels, receptors, or enzymes. To overcome these difficulties, some research groups used transgenic, knock-out and knock-in mice for NCX isoforms as the most straightforward and fruitful strategy to characterize the biological role exerted by each antiporter isoform. The present review will survey the techniques used to study the roles of NCXs and the current knowledge obtained from these genetic modified mice focusing on the advantages obtained with these strategies in understanding the contribution exerted by each isoform.     

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.     

Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+).

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.     

Na+ entry via TRPC6 causes Ca2+ entry via NCX reversal in ATP stimulated smooth muscle cells

Reversal of the Na+/Ca2+ -exchanger (NCX) has been shown to mediate Ca2+ influx during activation of G-protein linked receptors. Functional coupling between the reverse-mode NCX and the canonical transient receptor potential channels (TRPCs) has been proposed to mediate Ca2+ influx in HEK-293 cells overexpressing TRPC3. In this communication we present evidence for similar functional coupling of NCX to endogenously expressed TRPC6 in rat aorta smooth muscle cells. Selective inhibition of reverse-mode NCX with KB-R7943 and of non-selective cation-channels with SKF-96365 abolished Ca2+ influx in response to agonist stimulation (ATP). Expression of a dominant negative TRPC6 mutant also reduced the Ca2+ influx in proportion to its transfection efficiency. Calyculin A, which is known to disrupt the junctions of the plasma membrane and sarco/endoplasmic reticulum, increased global Na+ elevations and reduced stimulated Ca2+ influx. Together our data provide evidence that localized Na+ elevations are generated by TRPC6 and drive reversal of NCX to mediate Ca2+ influx.     

Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)

Three distinctmammalianNa⁺/Ca²⁺exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken adetailed functional comparison of these three exchangers. Eachexchanger was stably expressed at high levels in the plasma membranesof BHK cells. Na⁺/Ca²⁺exchange activity was assessed using three different complementary techniques: Na⁺ gradient-dependent⁴⁵Ca²⁺uptake into intact cells, Na⁺gradient-dependent⁴⁵Ca²⁺uptake into membrane vesicles isolated from the transfected cells, andexchange currents measured using giant patches of excised cellmembrane. Apparent affinities for the transported ionsNa⁺ andCa²⁺ were markedly similar for thethree exchangers at both membrane surfaces. Likewise, generally similarresponses to changes in pH, chymotrypsin treatment, and application ofvarious inhibitors were obtained. Depletion of cellular ATP inhibitedNCX1 and NCX2 but did not affect the activity of NCX3. Exchangeactivities of NCX1 and NCX3 were modestly increased by agents thatactivate protein kinases A and C. All exchangers were regulated byintracellular Ca²⁺. NCX1-inducedexchange currents were especially large in excised patches and, likethe native myocardial exchanger, were stimulated by ATP. Results may beinfluenced by our choice of expression system and specific splicevariants, but, overall, the three exchangers appear to have verysimilar properties.

     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.     

Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger.

Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.     

NCX3 is a major functional isoform of the sodium-calcium exchanger in osteoblasts

The calcium phosphate-based skeleton of vertebrates serves as the major reservoir for metabolically available calcium ions. The skeleton is formed by osteoblasts which first secrete a proteinaceous matrix and then provide Ca++ for the calcification process. The two calcium efflux ports found in most cells are the plasma membrane Ca-ATPase (PMCA) and the sodium-calcium exchanger (NCX). In osteoblasts, PMCA and NCX are located on opposing sides of the cell with NCX facing the mineralizing bone surface. Two isoforms of NCX have been identified in osteoblasts NCX1, and NCX3. The purpose of this study was to determine the extent to which each of the two NCX isoforms support delivery of Ca++ into sites of calcification and to discern if one could compensate for the other. SiRNA technology was used to knockdown each isoform separately in MC3T3-E1 osteoblasts. Osteoblasts in which either NCX1 or NCX3 was impaired were tested for Ca++ efflux using the Ca++ specific fluorophore, fluo-4, in a sodium-dependent calcium uptake assay adapted for image analysis. NCX3 was found to serve as a major contributor of Ca++ translocation out of osteoblasts into calcifying bone matrix. NCX1 had little to no involvement.     

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca²⁺]_i. However, the role of the three isoforms of the main Ca²⁺-extruding system, the Na⁺/Ca²⁺ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca²⁺]_i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca²⁺ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na⁺ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na⁺]_i significantly increased in cells overexpressing NCX1.4 as well as ER Ca²⁺ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca²⁺]_i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca²⁺ content and PI3K signaling.     

New perspectives for selective NCX activators in neurodegenerative diseases

The Na⁺/Ca²⁺ exchanger plays a relevant role in several neurological disorders, thus the pharmacological modulation of its isoforms might represent a promising strategy to ameliorate the course of some neurological pathologies including stroke, neonatal hypoxia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer Disease (AD), and spinal muscular atrophy (SMA). This review will summarize heterocyclic, peptidergic, genetic and epigenetic compounds activating or inhibiting the expression/activity of each NCX isoform. In addition, we will focus our attention on the development of new strategies aimed to ameliorate the pathophysiological conditions in which NCX isoform changes are found.     

TRPC6 and TRPC4 Heteromultimerization Mediates Store Depletion-Activated NCX1 Reversal in Proliferative Vascular Smooth Muscle Cells

Store depletion has been shown to induce Ca²⁺ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na⁺ was followed by a significant increase of cytosolic Ca²⁺ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca²⁺ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na⁺ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4–TRPC6 heteromultimerization linked Ca²⁺ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.     

Genetic manipulation of cardiac Na+/Ca2+ exchange expression

The Na+/Ca2+ exchanger (NCX) is the primary Ca2+ extrusion mechanism in cardiomyocytes. To further investigate the role of NCX in excitation-contraction coupling and Ca2+ homeostasis, we created murine models with altered expression levels of NCX. Homozygous overexpression of NCX resulted in mild cardiac hypertrophy. Decline of the Ca2+ transient and relaxation of contraction were increased and the reverse mode of NCX was augmented. Overexpression also led to a higher susceptibility to ischemia-reperfusion injury and to a greater ability of NCX to trigger Ca2+-induced Ca2+ release. Furthermore, an increase in peak L-type Ca2+ current was observed suggesting a direct influence of NCX on L-type Ca2+ current. Whereas global knockout of NCX led to prenatal death, a recently generated cardiac-specific NCX knockout mouse was viable with surprisingly normal contractile properties. Expression levels of other Ca2+-handling proteins were not altered. Ca2+ influx in these animals is limited by a decrease of peak L-type Ca2+ current. An alternative Ca2+ efflux mechanism, presumably the plasma membrane Ca2+-ATPase, is sufficient to maintain Ca2+-homeostasis in the NCX knockout mice.