| 2009 |
NCLX (encoded by SLC8B1/FLJ22233) is the mitochondrial Na+/Ca2+ exchanger responsible for Na+-dependent Ca2+ efflux from the mitochondrial matrix. It localizes to mitochondrial cristae, and its overexpression enhances mitochondrial Na+-dependent Ca2+ efflux while siRNA silencing reduces it. A catalytically inactive NCLX mutant blocks mitochondrial Ca2+ exchange. The exchanger exhibits Li+ dependence and sensitivity to CGP-37157, hallmarks of mitochondrial Na+-dependent Ca2+ efflux. |
Ca2+ and Na+ fluorescent imaging, siRNA knockdown, overexpression rescue, catalytically inactive mutant, immunofluorescence/fractionation showing cristae localization |
Proceedings of the National Academy of Sciences of the United States of America |
High |
20018762
|
| 2003 |
NCLX (NCKX6) exists in two alternatively spliced isoforms: the short isoform localizes to the plasma membrane and exhibits K+-dependent Na+/Ca2+ exchange activity in HEK-293 cells, whereas the full-length isoform is retained in the endoplasmic reticulum and does not mediate exchange. The short isoform is also present at the cardiac sarcolemmal membrane in native tissue. |
cDNA cloning, digital Ca2+ imaging (fura-2) in transfected HEK-293 cells, immunofluorescence with NCKX6-specific antibody in native cardiac tissue |
The Journal of biological chemistry |
High |
14625281
|
| 2006 |
Single α-repeat domain constructs (α1 or α2) of NCLX each independently mediate electrogenic Na+/Ca2+ exchange, but oligomerization (dimer or trimer) is required for full activity, as shown by dominant-negative co-expression and immunoprecipitation demonstrating physical interaction between constructs. An α2-S273T point mutation abolishes transport activity. |
Patch-clamp electrophysiology, fluorescence Ca2+ transport assays, dominant-negative co-expression dose-dependence analysis, immunoprecipitation in HEK-293 cells |
Biochemistry |
High |
17002286
|
| 2014 |
NCLX, but not LETM1, mediates Ca2+ extrusion from mitochondria in HeLa cells. NCLX overexpression enhances the rate of mitochondrial Ca2+ efflux and abolishes Ca2+-driven NAD(P)H production increases and matrix redox reduction, effects reversed by CGP37157. LETM1 overexpression has no impact on Ca2+ extrusion. |
Genetically encoded mitochondrial Ca2+ indicator, roGFP redox probe, NAD(P)H autofluorescence, NCLX/LETM1 overexpression, CGP37157 pharmacology in HeLa cells |
The Journal of biological chemistry |
High |
24898248
|
| 2015 |
PKA phosphorylates NCLX at serine 258, rescuing its activity in PINK1-deficient neurons. Loss of PINK1 inhibits NCLX-mediated mitochondrial Ca2+ extrusion; PKA pathway activation restores it. A phosphomimetic mutant NCLX(S258D) prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, enhancing neuronal survival. |
PKA pathway activation/inhibition, phosphomimetic and phospho-null mutagenesis (S258D), Ca2+ imaging in PINK1 KO neurons, mitochondrial membrane potential measurements, neuronal survival assays |
Cell reports |
High |
26440884
|
| 2017 |
Specific residues in the NCLX transport site confer selectivity for Li+ versus Na+ in Ca2+ exchange. Mutations N149A, P152A, D153A, N467Q, S468T, and G494S reduce Na+/Ca2+ but not Li+/Ca2+ exchange, while D471A reduces Li+/Ca2+ but not Na+/Ca2+ exchange. Simultaneous mutation of four putative Ca2+-binding residues completely abolishes both exchange activities. |
Site-directed mutagenesis, fluorescence monitoring of Li+- and Na+-dependent mitochondrial Ca2+ efflux in permeabilized and intact cells, molecular modeling on CAX_Af structure |
Biochimica et biophysica acta. Molecular cell research |
High |
28130126
|
| 2018 |
Mitochondrial membrane potential (ΔΨm) allosterically regulates NCLX activity: mild fluctuations in ΔΨm that do not affect Ca2+ influx strongly inhibit NCLX-mediated Ca2+ efflux. PKA phosphorylation at S258 (or S258D phosphomimetic) rescues NCLX from ΔΨm-driven allosteric inhibition. Additional NCLX residues that functionally interact with S258 to control ΔΨm sensitivity were identified. Glucose-driven ΔΨm changes in pancreatic β-cells control mitochondrial Ca2+ signaling primarily via NCLX. |
Mitochondrial Ca2+ transient monitoring during ΔΨm manipulation, phosphomimetic/phospho-null mutagenesis, NCLX mutant screening, pancreatic β-cell Ca2+ imaging with glucose stimulation |
Cell reports |
High |
30566870
|
| 2013 |
NCLX localizes exclusively to mitochondria in astrocytes (confirmed by organellar fractionation) and mediates mitochondrial Ca2+ extrusion. NCLX activity modulates store-operated Ca2+ entry (SOCE) at the plasma membrane. Silencing NCLX reduces Ca2+-dependent astrocyte functions including exocytotic glutamate release, in vitro wound closure, and proliferation, without affecting Ca2+ wave propagation. |
Organellar fractionation/immunoblot, pericam-based mitochondrial Ca2+ imaging, siRNA knockdown, CGP37157 pharmacology, glutamate release assay, wound-healing assay, proliferation assay in astrocytes |
The Journal of neuroscience |
High |
23616530
|
| 2013 |
NCLX knockdown in HL-1 cardiomyocytes markedly prolongs the cycle length of spontaneous Ca2+ oscillations and action potential generation, slows upstrokes, and compromises sarcoplasmic reticulum (SR) Ca2+ handling. Mathematical modeling shows that blocking mitochondrial NCX reduces SR Ca2+ content, slowing spontaneous SR Ca2+ leak that triggers automaticity. |
siRNA knockdown, Ca2+ oscillation and action potential imaging, mathematical modeling of HL-1 cardiomyocyte electrophysiology |
Scientific reports |
Medium |
24067497
|
| 2016 |
NCLX regulates B lymphocyte chemotaxis: inhibition or silencing of NCLX increases random migration and suppresses chemotactic response to CXCL12. NCLX knockdown elevates basal cytosolic Ca2+ and prevents CXCL12-induced Ca2+ increase, impairing Rac1 activation and F-actin polymerization. NCLX is required for CXCL12-induced enhancement of mitochondrial polarization. T lymphocyte chemotaxis is unaffected by NCLX inhibition, indicating B-cell specificity. |
siRNA knockdown, CGP-37157 pharmacological inhibition, chemotaxis/migration assays, cytosolic Ca2+ imaging, mitochondrial polarization measurement, Rac1 activation assay, F-actin staining in A20, DT40, and primary spleen B lymphocytes |
Scientific reports |
High |
27328625
|
| 2017 |
The C. elegans NCLX ortholog NCX-9 functions in hypodermal seam cells to enable axon guidance: ncx-9 mutants exhibit defects in stereotyped left/right axon guidance in the GABAergic motor neuron circuit. NCX-9 acts within a LON-2/heparan sulfate and UNC-6/netrin-mediated, RAC-dependent signaling pathway. In vitro physiology confirmed NCX-9 mediates Ca2+ exchange at the mitochondrion. |
C. elegans genetics (ncx-9 loss-of-function), epistasis analysis with LON-2, UNC-6, RAC pathway components, in vitro Ca2+ exchange physiology |
The Journal of biological chemistry |
Medium |
28196860
|
| 2020 |
Adrenergic stimulation of brown adipose tissue (BAT) activates PKA-dependent mitochondrial Ca2+ extrusion via NCLX. NCLX-null brown adipocytes show profound mitochondrial Ca2+ overload and impaired uncoupled respiration upon adrenergic stimulation. NCLX deletion causes mitochondrial permeability transition pore (mPTP) opening, mitochondrial swelling and cell death in BAT. mPTP inhibitors rescue mitochondrial function and thermogenesis in NCLX-null BAT while Ca2+ overload persists, placing NCLX upstream of mPTP in the adrenergic pathway. |
NCLX knockout mice, Ca2+ imaging, respiration measurements, mPTP inhibitor treatment, PET imaging, VO2 measurements, immunohistochemistry |
Nature communications |
High |
32620768
|
| 2020 |
NCLX downregulation causes mitochondrial Ca2+ overload, increased mitochondrial ROS, and activation of HIF1α signaling, which drives metastatic spread of colorectal cancer cells. NCLX loss leads to decreased cell-cycle gene expression and reduced tumor size in xenograft models, but promotes epithelial-to-mesenchymal transition, chemoresistance, and metastasis through mtCa2+ overload-ROS-HIF1α axis. |
NCLX knockdown/knockout in xenograft and spontaneous CRC mouse models, mitochondrial Ca2+ and ROS measurements, HIF1α pathway analysis, gene expression profiling |
eLife |
High |
32914752
|
| 2022 |
PDE2 inhibition enhances mitochondrial Ca2+ efflux via NCLX by increasing mitochondrial cAMP and promoting PKA-dependent phosphorylation of NCLX. This pathway protects hippocampal neurons against excitotoxic insults in an NCLX-dependent manner. Administration of PDE2 inhibitor Bay 60-7550 enhances new object recognition in wild-type but not NCLX knockout mice, demonstrating NCLX dependence of this cognitive effect. |
PDE2 inhibitor treatment, mitochondrial Ca2+ fluorescent monitoring, NCLX KO mice, new object recognition behavioral assay, PKA phosphorylation analysis |
Cell reports |
High |
36476859
|
| 2022 |
CKII phosphorylates NCLX at serine 271 to activate Ca2+ transport. CKII inhibitors suppress NCLX-dependent Ca2+ transport in neuronal cells; phosphomimetic S271D constitutively activates NCLX and renders it insensitive to CKII inhibition and ΔΨm-driven allosteric inhibition, while S271A blocks activity. NCLX is required for CKII-dependent distal relocation of axon initial segment (AIS) Na+ and Kv7 channels that underlies homeostatic axonal plasticity in hippocampal neurons. |
CKII inhibitor treatment, phosphomimetic/phospho-null mutagenesis (S271D/S271A), Ca2+ transport assays in SH-SY5Y and primary hippocampal neurons, NCLX-KO neurons, AIS channel localization imaging |
Cells |
High |
36552754
|
| 2022 |
NCLX is spatially and functionally coupled to SERCA at the sarcoplasmic reticulum-mitochondria interface in cardiomyocytes. Bimolecular fluorescence complementation shows NCLX is in close proximity to all four SERCA isoforms; super-resolution immunofluorescence shows higher co-localization of endogenous NCLX with SERCA2 than with ryanodine receptor or Na+/K+-ATPase. Mathematical modeling incorporating NCLX-SERCA spatial coupling reproduces NCLX inhibition-mediated modulations of SR Ca2+ reuptake. |
Bimolecular fluorescence complementation (BiFC) in HEK-293, super-resolution immunofluorescence in isolated ventricular myocytes, co-localization coefficient analysis, mathematical modeling |
International journal of molecular sciences |
Medium |
35887296
|
| 2022 |
NCLX is required for NCLX-dependent mitochondrial Ca2+ extrusion in airway smooth muscle (ASM), preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry (SOCE) and Ca2+/calmodulin-dependent kinase II (CaMKII) activation, which are required for ASM cell proliferation and migration. Smooth muscle-specific NCLX KO mice are protected against airway remodeling, fibrosis, and hyperresponsiveness in an experimental asthma model. |
Mitochondrial Ca2+-sensitive dyes, NCLX KO (smooth muscle-specific), RNA-Seq, CaMKII activation assays, small animal respiratory mechanics, immunohistochemistry in asthma mouse model |
The Journal of biological chemistry |
High |
35841929
|
| 2022 |
NCLX knockdown in hippocampal neurons and glia exacerbates mitochondrial Ca2+ dysregulation, mitochondrial membrane potential breakdown, and ROS generation during excitotoxic stimulation. NCLX-depleted neurons undergo cell death under basal conditions and during synaptic activity. In vivo NCLX knockdown in hippocampal CA1 causes substantial neurodegeneration and astrodegeneration. |
shRNA-mediated knockdown in primary hippocampal cultures and in vivo (CA1), mitochondrial Ca2+ imaging, ΔΨm measurement, ROS detection, cell death quantification during synaptic activity |
The Journal of biological chemistry |
High |
34942149
|
| 2022 |
Mitochondrial Lon protease interacts with and activates NCLX, and NCLX expression is dependent on Lon levels. In the context of cisplatin resistance, Lon-induced NCLX activation promotes Ca2+ release from mitochondria into the cytosol, activating the PYK2-SRC-STAT3-IL-6 pathway and conferring cisplatin resistance. |
Co-immunoprecipitation (Lon-NCLX interaction), NCLX inhibition, overexpression and knockdown of Lon and NCLX, mitochondrial and cytosolic Ca2+ measurements, in vitro and in vivo xenograft models |
Cell death & disease |
Medium |
35296653
|
| 2023 |
NLRP14 maintains NCLX protein stability by interacting with the NCLX intrinsically disordered regions (IDRs) domain and regulating K27-linked ubiquitination of NCLX. Loss of NLRP14 (or UHRF1) in oocytes leads to decreased NCLX levels, disrupted Ca2+ oscillations, and developmental arrest, which can be rescued by spindle transfer (cytoplasm substitution). |
Proteomics, co-immunoprecipitation (NLRP14-NCLX interaction), ubiquitination assay (K27-linked), Nlrp14 knockout mouse oocytes, spindle transfer rescue, Ca2+ oscillation imaging |
Advanced science |
High |
37493331
|
| 2023 |
Neuronal deletion of NCLX (Slc8b1) in mice results in age-dependent cognitive decline, moderate amyloid deposition, mild tau pathology, synaptic remodeling, and cell death, demonstrating that loss of NCLX-dependent mitochondrial Ca2+ efflux alone is sufficient to induce Alzheimer's disease-like pathology without pre-existing amyloid or tau pathology. |
Neuron-specific Slc8b1 conditional knockout mice, spatial and cued recall memory behavioral tests, amyloid and tau pathology immunohistochemistry, synaptic marker analysis |
iScience |
High |
36936788
|
| 2024 |
TMEM65, a mitochondrial inner membrane protein, is an NCLX binding partner that enhances Na+-dependent mitochondrial Ca2+ efflux. TMEM65 is required for Na+-dependent mCa2+ efflux; loss of TMEM65 promotes mCa2+ overload in heart and skeletal muscle and impairs cardiac and neuromuscular function. NCLX inhibition or deletion abolishes the TMEM65-dependent increase in mCa2+ efflux, establishing TMEM65 as an obligate regulator of NCLX. |
Proximity biotinylation proteomics (BioID), pharmacological NCLX inhibition, genetic NCLX deletion, TMEM65 KD in mice (cardiac and skeletal muscle), mCa2+ efflux assays, cardiac and neuromuscular function measurements |
Nature metabolism |
High |
37873405 40200126
|
| 2024 |
NCLX activity is required for HIF-α subunit stabilization during hypoxia and for HIF-1-dependent transcriptional activity. Using pharmacological NCLX inhibition and siRNA knockdown, NCLX was shown to be necessary for HIF-1α stabilization, linking NCLX-mediated mitochondrial ROS regulation to medium-term hypoxia gene expression responses. |
NCLX inhibitor (CGP-37157), siRNA knockdown, HIF-α stabilization assay, HIF-1-dependent transcriptional reporter assay under hypoxic conditions |
Redox biology |
Medium |
39341036
|
| 2024 |
Mfn2 (mitochondrial outer membrane fusion protein) physically interacts with NCLX and promotes NCLX-dependent mitochondrial Ca2+ release. Mfn2-NCLX interaction is strengthened by mitochondrial ROS and requires SLC25A46. ROS-induced NCLX-dependent Ca2+ release promotes mitophagy through NEDD4-1 (a Ca2+-responsive E3 ubiquitin ligase), independently of Mfn2-driven mitochondrial fission. |
Co-immunoprecipitation, proximity ligation assay, CETSA (thermal shift showing PXA targets Mfn2), NCLX KO and Mfn2 KO, pharmacological NCLX inhibition (CGP37157), Ca2+ imaging, mitophagy assays |
bioRxivpreprint |
Medium |
39149365
|
| 2024 |
NCLX regulates basal and starvation-induced autophagy through Ca2+ signaling. Conditions stimulating autophagy upregulate NCLX expression in hepatic tissue. NCLX knockdown or acute inhibition by CGP37157 impairs FIP200 puncta formation and downstream autophagosome biogenesis. NCLX inhibition reduces cytosolic Ca2+ levels, and intracellular Ca2+ chelation suppresses autophagy without additive effect on NCLX inhibition, demonstrating NCLX controls autophagy by modulating Ca2+ signaling. |
NCLX knockdown, CGP37157 pharmacological inhibition, FIP200 puncta assay, autophagosome biogenesis imaging, cytosolic Ca2+ measurement, Ca2+ chelation (BAPTA) non-additivity test in hepatic cells and in vivo caloric restriction |
FASEB journal |
Medium |
38315457
|
| 2026 |
Cryo-EM structures of rat NCLX in cytosolic-facing occluded and open states reveal a central transmembrane module of 10 helices organized as two inverted halves, with two α-repeats forming a central ion-binding pocket. Peripheral TMs 1 and 6 mediate alternative access. NCLX retains the canonical Ca2+-binding site but lacks key Na+-binding residues of NCX family members, indicating it functions as a non-selective cation/Ca2+ exchanger. Cell-based Ca2+ uptake assays confirm slower Ca2+ exchange than NCX and utilization of Na+, K+, Li+, and potentially protons as counterions. |
Cryo-EM structure determination (occluded and open states), cell-based Ca2+ uptake assays, structural comparison to NCX family |
bioRxivpreprint |
High |
41659638
|
| 2015 |
NCLX is present at the plasma membrane of rat INS-1 insulinoma cells (confirmed by immunocytochemistry and cell surface biotinylation) and mediates Na+-dependent Ca2+ movement across the plasma membrane. Plasmalemmal NCLX-mediated Ca2+ clearance is required specifically for sustained exocytosis (not initial exocytosis) and for maintaining mitochondrial membrane potential during repetitive stimulation. |
Immunocytochemistry, cell surface biotinylation, siRNA knockdown, capacitance measurement of exocytosis, mitochondrial membrane potential (ΔΨ) assay in INS-1 cells |
Pflugers Archiv |
Medium |
26100674
|