SHOC2 and CRAF Mediate ERK1/2 Reactivation in Mutant NRAS-mediated Resistance to RAF Inhibitor

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF^V600E melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF^V600E/NRAS^Q61K co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF^V600 cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF^V600E/NRAS^Q61K cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF^V600E/NRAS^Q61K cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

C57BL/6 congenic mouse NRASQ61K melanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo

RAS is frequently mutated in various tumors and known to be difficult to target. NRAS^Q61K/R are the second most frequent mutations found in human skin melanoma after BRAF^V600E. Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRAS^Q61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRAS^Q61K mouse melanoma that can be used in vitro and in vivo in an immune‐competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRAS^Q61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Proximity of Na+–Ca2+-exchanger and sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle: fluorescence microscopy

Pig coronary artery smooth muscle expresses the Na⁺–Ca²⁺-exchanger NCX1 and the sarco/endoplasmic reticulum (SER) Ca²⁺ pump SERCA2. NCX has been proposed to play a role in refilling the SER Ca²⁺ pool. Caveolae may also direct Ca²⁺ traffic during cell signaling. Here, we use immunofluorescence microscopy to determine if there is proximity between NCX1, SERCA2, and the caveolar protein caveolin-1. Stacks of images of cell surface domains were analyzed. Image stacks for one protein were analyzed for overlap with another protein, with and without randomization or image shifting. Within the resolution of light microscopy, there is significant overlap in the distributions of NCX1, SERCA2, and caveolin-1 but the three proteins are not always co-localized. The proximity between NCX1, SERCA2 is consistent with the assertion that NCX may supply Ca²⁺ for refilling the SER but this relationship is only partial. Similarly, caveolae may direct traffic in some Ca²⁺ signaling pathways but not others.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na⁺/Ca²⁺ exchanger (NCX) operating in the Ca²⁺-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca²⁺ influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na⁺-K⁺-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans'' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

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.     

The Na+/Ca2+ exchangers in demyelinating diseases

Intracellular [Na⁺]_i and [Ca²⁺]_i imbalance significantly contribute to neuro-axonal dysfunctions and maladaptive myelin repair or remyelination failure in chronic inflammatory demyelinating diseases such as multiple sclerosis. Progress in recent years has led to significant advances in understanding how [Ca²⁺]_i signaling network drive degeneration or remyelination of demyelinated axons.The Na⁺/Ca²⁺ exchangers (NCXs), a transmembrane protein family including three members encoded by ncx1, ncx2, and ncx3 genes, are emerging important regulators of [Na⁺]_i and [Ca²⁺]_i both in neurons and glial cells. Here we review recent advance highlighting the role of NCX exchangers in axons and myelin-forming cells, i.e. oligodendrocytes, which represent the major targets of the aberrant inflammatory attack in multiple sclerosis. The contribution of NCX subtypes to axonal pathology and myelin synthesis will be discussed. Although a definitive understanding of mechanisms regulating axonal pathology and remyelination failure in chronic demyelinating diseases is still lacking and requires further investigation, current knowledge suggest that NCX activity plays a crucial role in these processes. Defining the relative contributions of each NCX transporter in axon pathology and myelinating glia will constitute not only a major advance in understanding in detail the intricate mechanism of neurodegeneration and remyelination failure in demyelinating diseases but also will help to identify neuroprotective or remyelinating strategies targeting selective NCX exchangers as a means of treating MS.     

Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

Roles of Two Ca2+-binding Domains in Regulation of the Cardiac Na+-Ca2+ Exchanger

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.     

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca²⁺ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca²⁺ signals to trigger each of these independently is unknown. Other cells create local Ca²⁺ signals to activate Ca²⁺-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca²⁺ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca²⁺ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca²⁺ to isolate a cytosolic Ca²⁺ nanodomain created by Ca²⁺ release from the DTS. In the absence of any detectable rise in global cytosolic Ca²⁺ concentration, thrombin stimulation triggered Na⁺/Ca²⁺ exchanger (NCX)-dependent Ca²⁺ removal into the extracellular space, as well as Ca²⁺-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca²⁺ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca²⁺ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca²⁺ nanodomain contained within the MC that can control shape change independently of platelet aggregation.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

NCX activity generates spontaneous Ca2+ oscillations in the astrocytic leaflet microdomain

The synergy between synaptic Glu release and astrocytic Glu-Na⁺ symport is essential to the signalling function of the tripartite synapse. Here we used kinetic data of astrocytic Glu transporters (EAAT) and the Na⁺/Ca²⁺ exchanger (NCX) to simulate Glu release, Glu uptake and subsequent Na⁺ and Ca²⁺ dynamics in the astrocytic leaflet microdomain following single release event. Model simulations show that Glu-Na⁺ symport differently affect intracellular [Na⁺] in synapses with different extent of astrocytic coverage. Surprisingly, NCX activity alone has been shown to generate markedly stable, spontaneous Ca²⁺ oscillation in the astrocytic leaflet. These on-going oscillations appear when NCX operates either in the forward or reverse direction. We conjecture that intrinsic NCX activity may play a prominent role in the generation of astrocytic Ca²⁺ oscillations.     

Structure and Functional Analysis of a Ca2+ Sensor Mutant of the Na+/Ca2+ Exchanger

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 contraction/relaxation relies upon Ca²⁺ fluxes across the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca²⁺ influx (primarily through L-type Ca²⁺ channels) triggers the release of additional Ca²⁺ from the sarcoplasmic reticulum (SR),⁴ resulting in cardiomyocyte contraction. Removal of cytosolic Ca²⁺ by reuptake into the SR (through the SR Ca²⁺-ATPase) and expulsion from the cell (primarily through the Na⁺/Ca²⁺ exchanger, NCX1.1) results in relaxation (1). Altered Ca²⁺ cycling is observed in a number of pathophysiological situations including ischemia, hypertrophy, and heart failure (2). Understanding the function and regulation of NCX1.1 is thus of fundamental importance to understand cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na⁺ gradient to extrude Ca²⁺ in a ratio of three Na⁺ ions to one Ca²⁺ ion (3). In addition to transporting both Na⁺ and Ca²⁺, NCX1.1 is also strongly regulated by these two ions. Intracellular Na⁺ can induce NCX1.1 to enter an inactivated state, whereas Ca²⁺ bound to regulatory sites removes Na⁺-dependent inactivation and also activates Na⁺/Ca²⁺ exchange (3). These regulatory sites are located on a large cytoplasmic loop (∼500 residues located between transmembrane helices V and VI) containing two calcium binding domains (CBD1 and CBD2), which sense cytosolic Ca²⁺ levels. We have previously shown that Ca²⁺ binding to the primary site in CBD2 is required for full exchange regulation (4); CBD1, however, is a site of higher affinity and appears to dominate the activation of exchange activity by Ca²⁺.Both CBDs have an immunoglobulin fold formed from two antiparallel β sheets generating a β sandwich with a differing number of Ca²⁺ ions coordinated at the tip of the domain (4, 5). CBD1 binds four Ca²⁺ ions, whereas CBD2 binds only two Ca²⁺ ions. An initial NMR study revealed a local unfolding of the upper portion of CBD1 upon release of Ca²⁺ (6). In contrast, CBD2 did not display an unfolding response upon Ca²⁺ removal. A comparative analysis between CBDs revealed a difference in charge at residues in equivalent positions near the Ca²⁺ coordination site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of CBD1 upon Ca²⁺ removal was alleviated by reversing the charge of the acidic residue (E454K) involved in Ca²⁺ coordination (6). Previously, we solved the structures of the Ca²⁺-bound and -free conformations of CBD2 and revealed a charge compensation mechanism involving Lys-585 (4). The positively charged lysine residue assumes the position of one of the Ca²⁺ ions upon Ca²⁺ depletion, permitting CBD2 to retain its overall fold (4). A similar phenomenon is predicted to take place in E454K-CBD1 mutant. In addition, Hilge et al. (6) showed that the E454K mutation of CBD1 decreases Ca²⁺ affinity to a level similar to that of CBD2 and suggested that the E454K mutation would cause the loss of primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR study (7) of CBD1 under more physiologically relevant conditions revealed no significant alteration in tertiary structure in the absence of Ca²⁺. It was hypothesized that Ca²⁺ binding induces localized conformational and dynamic changes involving several of the binding site residues. To clarify this issue, we solved the crystal structure of the E454K-CBD1 mutant and examined the functional effects of different CBD1 mutations in the full-length NCX1.1. The results indicate that charge compensation is indeed provided by the residue Lys-454 to replace one Ca²⁺, whereas the overall E454K-CBD1 structure is only slightly perturbed. The charge compensation, however, has no impact on Ca²⁺ regulation of NCX1.1.     

Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization

The mechanism whereby events in and around the catalytic site/head of Ca²⁺-ATPase effect Ca²⁺ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca²⁺ by taking advantage of its rapid occlusion upon formation of E1PCa₂ and of stabilization afforded by a high concentration of Ca²⁺. The assay is applicable to minute amounts of Ca²⁺-ATPase expressed in COS-1 cells. It was validated by measuring the Ca²⁺ binding properties of unphosphorylated Ca²⁺-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca²⁺ release process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. In the wild type, Ca²⁺ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa₂ → E2PCa₂) followed by very rapid Ca²⁺ release. In contrast, with alanine mutants of Leu¹¹⁹ and Tyr¹²² on the cytoplasmic part of the second transmembrane helix (M2) and Ile¹⁷⁹ on the A domain, Ca²⁺ release in 10 μm Ca²⁺ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca²⁺. The results suggest that these residues function in Ca²⁺ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca²⁺ release.