RESUMO
Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.
Assuntos
Cálcio , Magnésio , Trocador de Sódio e Cálcio , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Trocador de Sódio e Cálcio/genética , Magnésio/metabolismo , Cálcio/metabolismo , Sítios de Ligação , Humanos , Regulação Alostérica , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios ProteicosRESUMO
Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.
Assuntos
Detergentes , Conformação Proteica , Trocador de Sódio e Cálcio , Detergentes/química , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Trocador de Sódio e Cálcio/genética , Íons/química , Nanoestruturas/química , Solubilidade , Animais , Espectroscopia de Ressonância Magnética/métodosRESUMO
The cardiac Na+/Ca2+ Exchanger (NCX1) controls transmembrane calcium flux in numerous tissues. The only reversible post-translational modification established to regulate NCX1 is palmitoylation, which alters the ability of the exchanger to inactivate. Palmitoylation creates a binding site for the endogenous XIP domain, a region of the NCX1 intracellular loop established to inactivate NCX1. The binding site created by NCX1 palmitoylation sensitizes the transporter to XIP. Herein we summarize our recent knowledge on NCX1 palmitoylation and its association with cardiac pathologies, and discuss these findings in the light of the recent cryo-EM structures of human NCX1.
Assuntos
Lipoilação , Processamento de Proteína Pós-Traducional , Trocador de Sódio e Cálcio , Trocador de Sódio e Cálcio/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/química , Humanos , Animais , Sítios de Ligação , Cálcio/metabolismo , Miocárdio/metabolismoRESUMO
Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.
Assuntos
Mamíferos , Trocador de Sódio e Cálcio , Animais , Estrutura Secundária de Proteína , Trocador de Sódio e Cálcio/químicaRESUMO
Sodium-calcium exchanger proteins influence calcium homeostasis in many cell types and participate in a wide range of physiological and pathological processes. Here, we elucidate the cryo-EM structure of the human Na+/Ca2+ exchanger NCX1.3 in the presence of a specific inhibitor, SEA0400. Conserved ion-coordinating residues are exposed on the cytoplasmic face of NCX1.3, indicating that the observed structure is stabilized in an inward-facing conformation. We show how regulatory calcium-binding domains (CBDs) assemble with the ion-translocation transmembrane domain (TMD). The exchanger-inhibitory peptide (XIP) is trapped within a groove between the TMD and CBD2 and predicted to clash with gating helices TMs1/6 at the outward-facing state, thus hindering conformational transition and promoting inactivation of the transporter. A bound SEA0400 molecule stiffens helix TM2ab and affects conformational rearrangements of TM2ab that are associated with the ion-exchange reaction, thus allosterically attenuating Ca2+-uptake activity of NCX1.3.
Assuntos
Cálcio , Trocador de Sódio e Cálcio , Humanos , Compostos de Anilina/farmacologia , Cálcio/metabolismo , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/químicaRESUMO
Calcium signaling is ubiquitous in nematode development from fertilization to cell specification to apoptosis. Calcium also regulates dauer entry in Caenorhabditis elegans, which corresponds to the infective stage of parasitic nematodes. In diverse parasites such as Trypanosoma cruzi and Toxoplasma gondii calcium has been shown to regulate host cell entry and egress, and perturbing calcium signaling represents a possible route to inhibit infection and parasitism in these species. Sodium calcium exchangers are considered the most important mechanism of calcium efflux, and our lab has previously characterized the sodium calcium exchanger gene family in C. elegans and studied the diversity of this family across a subset of specific nematode species. Here we build upon these data and explore sodium calcium exchangers across 108 species of nematodes. Our data reveal substantial differences in sodium calcium exchanger counts across the Phylum and detail expansions and contractions of specific exchanger subtypes within certain nematode clades. Finally, we also provide evidence for a role of sodium calcium exchangers in parasite activation by examining differentially expressed genes in non-activated versus activated infective stage larvae. Taken together our findings paint a heterogeneous picture of sodium calcium exchanger evolution across the Phylum Nematoda that may reflect unique adaptations to free-living and parasitic lifestyles.
Assuntos
Proteínas de Helminto/genética , Nematoides/genética , Trocador de Sódio e Cálcio/genética , Motivos de Aminoácidos , Animais , Caenorhabditis elegans/genética , Sinalização do Cálcio , Evolução Molecular , Estudo de Associação Genômica Ampla , Humanos , Nematoides/classificação , Nematoides/fisiologia , Infecções por Nematoides/parasitologia , Trocador de Sódio e Cálcio/químicaRESUMO
It has been over 10 years since SLC24A6/SLC8B1, coding the Na+/Ca2+/Li+ exchanger (NCLX), was identified as the gene responsible for mitochondrial Na+-Ca2+ exchange, a major Ca2+ efflux system in cardiac mitochondria. This molecular identification enabled us to determine structure-function relationships, as well as physiological/pathophysiological contributions, and our understandings have dramatically increased. In this review, we provide an overview of the recent achievements in relation to NCLX, focusing especially on its heart-specific characteristics, biophysical properties, and spatial distribution in cardiomyocytes, as well as in cardiac mitochondria. In addition, we discuss the roles of NCLX in cardiac functions under physiological and pathophysiological conditions-the generation of rhythmicity, the energy metabolism, the production of reactive oxygen species, and the opening of mitochondrial permeability transition pores.
Assuntos
Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Animais , Metabolismo Energético , Humanos , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Modelos Moleculares , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes.
Assuntos
Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sinalização do Cálcio , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Feminino , Hipocampo/metabolismo , Humanos , Técnicas In Vitro , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Proteínas Mitocondriais/deficiência , Plasticidade Neuronal , Neurônios/metabolismo , Linhagem , Mutação Puntual , Terminações Pré-Sinápticas/metabolismo , Trocador de Sódio e Cálcio/química , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1-5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a â¼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.
Assuntos
Antiporters/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Antiporters/genética , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Trocador de Sódio e Cálcio/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Sodium-calcium exchanger (NCX) 1 and 3, have been demonstrated to play a relevant role in controlling the intracellular homeostasis of sodium and calcium ions in physiological and patho-physiological conditions. While NCX1 and NCX3 knocking-down have been both implicated in brain ischemia, several aspects of the epigenetic regulation of these two antiporters transcription were not yet well characterized. In response to stroke, NCX1 and NCX3 transcriptional regulation occurs from specific promoter sequences. Several evidences have shown that the expression of NCX1 and NCX3 can be determined by epigenetic modifications, consisting in changes of the histone acetylation levels on their promoter sequences. An interesting issue is that histone modifications at the NCX1 and NCX3 promoters could be linked to neurodegeneration occurring after stroke. Therefore, identifying the epigenetic regulation at the NCX1 and NCX3 promoters could permit to identify new molecular targets that can open new strategies for stroke treatment. The current review reassumes the recent knowledge of histone modifications of NCX1 and NCX3 genes in brain in physiological and patho-physiological conditions.
Assuntos
Encéfalo/metabolismo , Epigênese Genética , Trocador de Sódio e Cálcio/genética , Transcrição Gênica , Animais , Humanos , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A precise temporal and spatial control of intracellular Ca2+ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca2+ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). NCX extrudes Ca2+ from the cell, balancing the Ca2+entering the cytoplasm during systole through L-type Ca2+ channels. In parallel, following SR Ca2+ release, SERCA activity replenishes the SR, reuptaking Ca2+ from the cytoplasm. The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca2+ potentiate NCX activity, while an increase in intracellular Na+ levels inhibits NCX via a mechanism known as Na+-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca2+, Na+ and H+ couple cell metabolism to Ca2+ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.
Assuntos
Citoplasma/metabolismo , Miocárdio/metabolismo , Prótons , Trocador de Sódio e Cálcio/metabolismo , Regulação Alostérica , Animais , Humanos , Concentração de Íons de Hidrogênio , Trocador de Sódio e Cálcio/químicaRESUMO
Selective recognition and transport of Na+ and Ca2+ ions by sodium-calcium exchanger (NCX) proteins is a primary prerequisite for Ca2+ signaling and homeostasis. Twelve ion-coordinating residues are highly conserved among NCXs, and distinct NCX orthologs contain two or three carboxylates, while sharing a common ion-exchange stoichiometry (3Na+ :1Ca2+ ). How these structural differences affect the ion-binding affinity, selectivity, and transport rates remains unclear. Here, the mutational effects of three carboxylates (E54, E213, and D240) were analyzed on the ion-exchange rates in the archaeal NCX from Methanococcus jannaschii and ion-induced structure-affinity changes were monitored by attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR). The D240N mutation elevated the ion-transport rates by twofold to threefold, meaning that the deprotonation of D240 is not essential for transport catalysis. In contrast, mutating E54 or E213 to A, D, N, or Q dramatically decreased the ion-transport rates. ATR-FTIR revealed high- and low-affinity binding of Na+ or Ca2+ with E54 and E213, but not with D240. These findings reveal distinct structure-affinity states at specific ion-binding sites in the inward-facing (IF) and outward-facing orientation. Collectively, two multidentate carboxylate counterparts (E54 and E213) play a critical role in determining the ion coordination/transport in prokaryotic and eukaryotic NCXs, whereas the ortholog substitutions in prokaryotes (aspartate) and eukaryotes (asparagine) at the 240 position affect the ion-transport rates differently (kcat ), probably due to the structural differences in the transition state.
Assuntos
Proteínas Arqueais/metabolismo , Cálcio/metabolismo , Methanocaldococcus/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Sítios de Ligação/genética , Ligação Competitiva , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Cinética , Methanocaldococcus/genética , Mutação , Ligação Proteica , Domínios Proteicos , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.
Assuntos
Lipoilação , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ativação do Canal Iônico , Modelos Biológicos , Miocárdio/metabolismo , Miocárdio/patologia , Trocador de Sódio e Cálcio/químicaRESUMO
K+-dependent Na+/Ca2+ exchanger proteins (NCKX1-5) of the SLC24 gene family play important roles in a wide range of biological processes including but not limited to rod and cone photoreceptor vision, olfaction, enamel formation and skin pigmentation. NCKX proteins are also widely expressed throughout the brain and NCKX2 and NCKX4 knockouts in mice have specific phenotypes. Here we review our work on structure-function relationships of NCKX proteins. We discuss membrane topology, domains critical to transport function, and residues critical to cation binding and transport function, all in the context of crystal structures that were obtained for the archaeal Na+/Ca2+ exchanger NCX_Mj.
Assuntos
Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Humanos , Modelos Moleculares , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
The solute carrier 8 (SLC8) family of sodium-calcium exchangers (NCXs) functions as an essential regulatory system that couples opposite fluxes of sodium and calcium ions across plasmalemmal membranes. NCXs, thereby, play key roles in maintaining an ion homeostasis that preserves cellular integrity. Hence, alterations in NCX expression and regulation have been found to lead to ionic imbalances that are often associated with intracellular calcium overload and cell death. On the other hand, intracellular calcium has been identified as a key driver for a multitude of downstream signaling events that are crucial for proper functioning of biological systems, thus highlighting the need for a tightly controlled balance. In the CNS, NCXs have been primarily characterized in the context of synaptic transmission and ischemic brain damage. However, a much broader picture is emerging. NCXs are expressed by virtually all cells of the CNS including oligodendrocytes (OLGs), the cells that generate the myelin sheath. With a growing appreciation of dynamic calcium signals in OLGs, NCXs are becoming increasingly recognized for their crucial roles in shaping OLG function under both physiological and pathophysiological conditions. In order to provide a current update, this review focuses on the importance of NCXs in cells of the OLG lineage. More specifically, it provides a brief introduction into plasmalemmal NCXs and their modes of activity, and it discusses the roles of OLG expressed NCXs in regulating CNS myelination and in contributing to CNS pathologies associated with detrimental effects on OLG lineage cells.
Assuntos
Homeostase/fisiologia , Oligodendroglia/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Animais , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Trocador de Sódio e Cálcio/químicaRESUMO
Structure-dynamic analysis of archaeal NCX (NCX_Mj) provided new insights into the underlying mechanisms of ion selectivity, ion-coupled alternating access, ion occlusion, and transport catalysis. This knowledge is relevant, not only for prokaryotic and eukaryotic NCXs, but also for other families belonging to the superfamily of Ca2+/CA antiporters. In parallel with the ion transport mechanisms, the structure-dynamic determinants of regulatory CBD1 and CBD2 domains have been resolved according to which the Ca2+-induced allosteric signal is decoded at the two-domain interface and "secondarily" modified by a splicing segment at CBD2. The exon-dependent combinations within the splicing segment control the number of Ca2+ binding sites (from zero to three) at CBD2, as well as the Ca2+ binding affinity and Ca2+ off-rates at both CBDs. The exon-dependent combinations specifically rigidify the local segments at CBDs, yielding the Ca2+-dependent activation (through Ca2+ binding to CBD1) and Ca2+-dependent alleviation of Na+-induced inactivation (through Ca2+ binding with CBD2). The exon-dependent synergistic interactions between CBDs characteristically differ in NCX1 and NCX3, thereby underscoring the physiological relevance of structure-controlled shaping of ion-dependent regulation in tissue-specific NCX variants. How the ion-dependent regulatory modules operate in conjunction with other regulators (PIP2, palmitoylation, XIP, among the others) of NCX is an open question that remains to be determined.
Assuntos
Edição de RNA , Trocador de Sódio e Cálcio/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Humanos , Transporte de Íons , Isoformas de Proteínas/metabolismo , Trocador de Sódio e Cálcio/química , Relação Estrutura-AtividadeRESUMO
All cells must control the activities of their ion channels and transporters to maintain physiologically appropriate gradients of solutes and ions. The complexity of underlying regulatory mechanisms is staggering, as exemplified by insulin regulation of transporter trafficking. Simpler strategies occur in single-cell organisms, where subsets of transporters act as solute sensors to regulate expression of their active homologues. This Viewpoint highlights still simpler mechanisms by which Na transporters use their own transport sites as sensors for regulation. The underlying principle is inherent to Na/K pumps in which aspartate phosphorylation and dephosphorylation are controlled by occupation of transport sites for Na and K, respectively. By this same principle, Na binding to transport sites can control intrinsic inactivation reactions that are in turn modified by extrinsic signaling factors. Cardiac Na/Ca exchangers (NCX1s) and Na/K pumps are the best examples. Inactivation of NCX1 occurs when cytoplasmic Na sites are fully occupied and is regulated by lipid signaling. Inactivation of cardiac Na/K pumps occurs when cytoplasmic Na-binding sites are not fully occupied, and inactivation is in turn regulated by Ca signaling. Potentially, Na/H exchangers (NHEs) and epithelial Na channels (ENaCs) are regulated similarly. Extracellular protons and cytoplasmic Na ions oppose secondary activation of NHEs by cytoplasmic protons. ENaCs undergo inactivation as cytoplasmic Na rises, and small diffusible molecules of an unidentified nature are likely involved. Multiple other ion channels have recently been shown to be regulated by transiting ions, thereby underscoring that ion permeation and channel gating need not be independent processes.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Canais Epiteliais de Sódio/química , Homeostase , Humanos , Transporte de Íons , Trocador de Sódio e Cálcio/química , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
NCKX5 is a bidirectional K+ -dependent Na+ -Ca2+ exchanger, which belongs to the SLC24A gene family. In particular, the A111T mutation of NCKX5 has been associated with reduced pigmentation in European populations. In contrast to other NCKX isoforms, which function in the plasma membrane (PM), NCKX5 has been shown to localize either in the trans-Golgi network (TGN) or in melanosomes. Moreover, sequences responsible for retaining its intracellular localization are unknown. This study addresses two major questions: (i) clarification of intracellular location of NCKX5 and (ii) identification of sequences that retain NCKX5 inside the cell. We designed a set of cDNA constructs representing NCKX5 loop deletion mutants and NCKX2-NCKX5 chimeras to address these two questions after expression in pigmented MNT1 cells. Our results show that NCKX5 is not a PM resident and is exclusively located in the TGN. Moreover, the large cytoplasmic loop is the determinant for retaining NCKX5 in the TGN.
Assuntos
Pigmentação , Potássio/farmacologia , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/metabolismo , Cálcio/metabolismo , Contagem de Células , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Mutação/genética , Pigmentação/efeitos dos fármacos , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-Atividade , Peixe-Zebra , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
The cell membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers control Ca2+ homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na+ and Ca2+, whereas NCLX can exchange Ca2+ with either Na+ or Li+. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (Kintâ¯=â¯0.8-1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kintâ¯=â¯0.1-0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the "Ca2+-site" (SCa) of NCLX_Mj binds Na+, Li+, or Ca2+, whereas one or more additional Na+/Li+ sites of NCLX_Mj are incompatible with the Na+ sites (Sext and Sint) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na+/Li+ (but not for Ca2+) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca2+ homeostasis in distinct sub-cellular compartments.
Assuntos
Transporte de Íons , Lítio/metabolismo , Proteínas Mutantes/química , Trocador de Sódio e Cálcio/química , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Cálcio/metabolismo , Membrana Celular , Homeostase , Proteínas de Membrana , Proteínas MitocondriaisRESUMO
Calcium is a key regulator of mitochondrial function under both normal and pathological conditions. The mechanisms linking metabolic activity to mitochondrial Ca2+ signaling remain elusive, however. Here, by monitoring mitochondrial Ca2+ transients while manipulating mitochondrial membrane potential (ΔΨm), we found that mild fluctuations in ΔΨm, which do not affect Ca2+ influx, are sufficient to strongly regulate NCLX, the major efflux pathway of Ca2+ from the mitochondria. Phosphorylation of NCLX or expression of phosphomimicking mutant (S258D) rescued NCLX activity from ΔΨm-driven allosteric inhibition. By screening ΔΨm sensitivity of NCLX mutants, we also identified amino acid residues that, through functional interaction with Ser258, control NCLX regulation. Finally, we find that glucose-driven ΔΨm changes in pancreatic ß-cells control mitochondrial Ca2+ signaling primarily via NCLX regulation. Our results identify a feedback control between metabolic activity and mitochondrial Ca2+ signaling and the "safety valve" NCLX phosphorylation that can rescue Ca2+ efflux in depolarized mitochondria.