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1.
Structure-dynamic and functional relationships in a Li+-transporting sodium‑calcium exchanger mutant
Moshe Giladi Su Youn Lee Bosmat Refaeli Reuben Hiller Ka Young Chung Daniel Khananshvili 《BBA》2019,1860(3):189-200
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 (Kint?=?0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kint?=?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. 相似文献
2.
《Channels (Austin, Tex.)》2013,7(5):322-328
The Na+-Ca2+ exchanger (NCX) links transmembrane movements of Ca2+ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca2+ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca2+ influx under certain conditions. Na+ and Ca2+ ions exert complex regulatory effects on NCX activity. Ca2+ binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na+ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca2+ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP2) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na+ concentrations also induce an inactivated state of the exchanger (Na+-dependent inactivation) that becomes dominant when cytosolic pH and PIP2 levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca2+ overload due to NCX-mediated Ca2+ influx during ischemia. 相似文献
3.
Na+/Ca2+ exchangers (NCXs) promote the extrusion of intracellular Ca2+ to terminate numerous Ca2+-mediated signaling processes. Ca2+ interaction at two Ca2+ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca2+ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca2+ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca2+ binding. The carboxylate residues that coordinate two Ca2+ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca2+ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca2+ binding site to change Ca2+ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca2+ regulatory behavior. 相似文献
4.
《Cell calcium》2020
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. 相似文献
5.
The P2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca2+]i due to Ca2+ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca2+ release was accompanied by a Ca2+- dependent regulatory activation of Na+/Ca2+ exchange activity: Addition of extracellular Ca2+ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca2+]i in Na+-free media (Li+ substitution) compared to Na+-containing media; no differences between Na+- and Li+-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na+ and Ca2+, the ATP-evoked rise in [Ca2+]i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca2+]i which eventually came to exceed the declining [Ca2+]i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba2+ influx, consistent with regulatory activation of the Na+/Ca2+ exchanger. The acceleration of Ba2+ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na+ or when the Ca2+ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca2+]i under Na+-free conditions, but did not inhibit the ATP-evoked stimulation of Ba2+ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca2+ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca2+ release from intracellular stores through regulatory activation by increased [Ca2+]i. In the absence of extracellular Ca2+, the stimulation of exchange activity is short-lived and follows the time course of the [Ca2+]i transient; in the presence of extracellular Ca2+, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca2+ entry. 相似文献
6.
《Biophysical journal》2021,120(17):3664-3675
Na+/Ca2+ exchangers (NCXs) are secondary active transporters that couple the translocation of Na+ with the transport of Ca2+ in the opposite direction. The exchanger is an essential Ca2+ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca2+-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca2+ sensor called CBD12. Binding of intracellular Ca2+ to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca2+-bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR (1H-15N residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca2+-bound state is less frequently sampled. These results are consistent with the view that Ca2+ binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs’ allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems. 相似文献
7.
《Cell calcium》2020
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. 相似文献
8.
《生物化学与生物物理学报:生物膜》2022,1864(1):183792
The Na+/Ca2+ exchangers (NCXs) modulate the Ca2+ signaling and homeostasis in health and disease.The transport cycle turnover rates (kcat) and the kcat/Km values of eukaryotic NCXs are ~104-times higher than those of prokaryotic NCXs.Three ion-coordinating residues (out of twelve) differ between eukaryotic NCXs and NCX_Mj.The replacement of three ion-coordinating residues in NCX_Mj does not increase kcat, probably due to the structural rigidity of NCX_Mj. Phospholipids and cholesterol increase (up to 10-fold) the transport rates in the cardiac NCX1.1, but not in NCX_Mj.A lipid environment can partially contribute to the huge kinetic variances among NCXs. 相似文献
9.
Satoshi Matsuoka Debora A. Nicoll Zhaoping He Kenneth D. Philipson 《The Journal of general physiology》1997,109(2):273-286
The cardiac sarcolemmal Na+-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A
large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A.
Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson.
1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has
an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties.
The major alteration was in a regulatory mechanism known as Na+-dependent inactivation. This inactivation is
manifested as a partial decay in outward Na+-Ca2+ exchange current after application of Na+ to the intracellular
surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was
markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na+ affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region
mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na+-dependent
inactivation. XIP-region mutants were all still regulated by Ca2+. However, the apparent affinity of the group 1 mutants for regulatory Ca2+ was decreased. The responses of all mutant exchangers to Ca2+ application or removal
were markedly accelerated. Na+-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement
of the exchanger into and out of the Na+-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+. 相似文献
10.
The role of intracellular sodium in the regulation of NMDA-receptor-mediated channel activity and toxicity 总被引:2,自引:0,他引:2
Yu XM 《Molecular neurobiology》2006,33(1):63-79
Sodium (Na+) is the major cation in extracellular space and, with its entry into cells, may act as a critical intracellular second messenger
that regulates many cellular functions. Through our investigations of mechanisms underlying the activity-dependent regulation
of N-methyl-d-aspartate (NMDA) receptors, we recently characterized intracellular Na+ as a possible signaling factor common to processes underlying the upregulation of NMDA receptors by non-NMDA glutamate channels,
voltage-gated Na+ channels, and remote NMDA receptors. Furthermore, although Ca2+ influx during the activation of NMDA receptors acts as a negative feedback mechanism that downregulates NMDA receptor activity,
Na+ influx provides an essential positive feedback mechanism to overcome Ca2+-induced inhibition, thereby potentiating both NMDA receptor activity and inward Ca2+ flow. NMDA receptors may be recruited to cause excitoxicity through a Na+-dependent mechanism. Therefore, the further characterization of mechanisms underlying the regulation of NMDA receptors by
intracellular Na+ is essential to understanding activity-dependent neuroplasticity in the nervous system. 相似文献
11.
《Cell calcium》2020
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. 相似文献
12.
New insights into the molecular and cellular workings of the cardiac Na+/Ca2+ exchanger 总被引:3,自引:0,他引:3
Hilgemann DW 《American journal of physiology. Cell physiology》2004,287(5):C1167-C1172
The cardiac Na+/Ca2+ exchanger (NCX1) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes, although the driving force for Ca2+ extrusion is quite small. To explain multiple recent results, it is useful to think of the exchanger as a slow Ca2+ buffer that can reverse its function multiple times during the excitation-contraction cycle (ECC). An article by the group of John Reeves brings new insights to this function by analyzing the role of regulatory domains of NCX1 that mediate its activation by a rise of cytoplasmic Ca2+. It was demonstrated that the gating reactions are operative just in the physiological range of Ca2+ changes, a few fold above resting Ca2+ level, and that they prevent the exchanger from damping out the influence of mechanisms that transiently increase Ca2+ levels. Furthermore, exchangers with deleted regulatory domains are shown to reduce resting Ca2+ to lower levels than achieved by wild-type exchangers. A study by the group of Kenneth Philipson demonstrated that the NCX1 regulatory domain can bind and respond to Ca2+ changes on the time scale of the ECC in rat myocytes. At the same time, studies of transgenic mice and NCX1 knockout mice generated by the Philipson group revealed that large changes of NCX1 activity have rather modest effects on ECC. Simple simulations predict these results very well: murine cardiac ECC is very sensitive to small changes of the Na+ gradient, very sensitive to changes of the sarcoplasmic reticulum Ca2+ pump activity, and very insensitive to changes of NCX1 activity. It is speculated that the NCX1 gating reactions not only regulate coupled 3Na+:1Ca2+ exchange but also control the exchangers Na+ leak function that generates background Na+ influx and depolarizing current in cardiac myocytes. excitation-contraction cycle 相似文献
13.
C.W McLaughlin 《生物化学与生物物理学报:生物膜》1973,323(2):285-296
Slicing and incubating rat liver caused a rapid Ca2+-independent exchange of K+ for Na+, followed by a Ca2+-dependent recovery. Freshly cut slices washed for 10 min in a Ca2+ medium containing equal concentrations of Na+ and K+ showed little replacement of K+ by Na+ during subsequent incubation in a normal medium. Changes in medium Ca2+ caused immediate changes in slice Na+ and K+, before any substantial change in slice Ca2+ and without altering gradients responsible for passive transfers of Na+ and K+. Ca2+ did not influence an ouabain-sensitive Na+ pump. It also appeared unlikely that Ca2+ was required for an ouabain-insensitive Na+ pump or for maintenance of intracellular structures concerned with K+ sorption, even if these mechanisms existed in the slices. Instead Ca2+ seemed to maintain the cell membrane relatively impermeable to Na+ and K+. An ouabain-sensitive Na+ pump not normally dependent on oxygen supply to the cells appeared to alter its activity according to the work required of it. Control of slice water content could not be attributed to the activity of this pump. 相似文献
14.
15.
Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster
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Alexander Omelchenko Christopher Dyck Mark Hnatowich John Buchko Debora A. Nicoll Kenneth D. Philipson Larry V. Hryshko 《The Journal of general physiology》1998,111(5):691-702
Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+
o exchanges for bath Na+
i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+
i affinities and current–voltage relationships, significant differences were observed in their Na+
i- and Ca2+
i-dependent regulatory properties. Both isoforms underwent Na+
i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+
i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+
i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+
i, but their responses to regulatory Ca2+
i differed markedly. For both isoforms, the application of cytoplasmic Ca2+
i led to a decrease in outward exchange currents. This negative regulation by Ca2+
i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+
i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca2+
i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca2+
i occurred by a direct mechanism and indirectly through effects on Na+
i-induced inactivation. Our results show that regulatory Ca2+
i decreases Na+
i-induced inactivation of CALX1.2, whereas it stabilizes the Na+
i-induced inactive state of CALX1.1. These effects of Ca2+
i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins. 相似文献
16.
The possible contribution of Na+-Ca2+ exchange to the triggering of Ca2+ release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the “trigger flux” of Ca2+ crossing the cell membrane in rabbit ventricular myocytes with Ca2+ release disabled pharmacologically. Under conditions that promote Ca2+ entry via Na+-Ca2+ exchange, internal [Na+] (10 mM), and positive membrane potential, the Ca2+ trigger flux (measured using a fluorescent Ca2+ indicator) was much greater than the Ca2+ flux through the L-type Ca2+ channel, indicating a significant contribution from Na+-Ca2+ exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca2+ channels was assessed by whole-cell patch-clamp recordings of Ca2+ current and complementary experiments in which internal [Na+] was reduced. However, Ca2+ entry via Na+-Ca2+ exchange measured in the absence of L-type Ca2+ current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca2+ channels increase [Ca2+] near Na+-Ca2+ exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca2+ release in heart cells under various conditions. 相似文献
17.
《Cell calcium》2020
It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca2+ signaling, and it is now generally recognized that control over Ca2+ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca2+ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca2+ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na+/Ca2+ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca2+ and Na+ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca2+ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease. 相似文献
18.
Maria Barthmes Jun Liao Youxing Jiang Andrea Brüggemann Christian Wahl-Schott 《The Journal of general physiology》2016,147(6):485-496
Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology. 相似文献
19.
Ádám Fekete Laura Franklin Takeshi Ikemoto Balázs Rózsa Balázs Lendvai E. Sylvester Vizi Tibor Zelles 《Journal of neurochemistry》2009,111(3):745-756
Although the role of Na+ in several aspects of Ca2+ regulation has already been shown, the exact mechanism of intracellular Ca2+ concentration ([Ca2+]i) increase resulting from an enhancement in the persistent, non‐inactivating Na+ current (INa,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na+ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na+ concentration ([Na+]i) and biphasic [Ca2+]i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca2+ response was tetrodotoxin‐ and extracellular Ca2+‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca2+]i rise was the net result of Ca2+ influx through voltage‐gated Ca2+ channels and mitochondrial Ca2+ sequestration. The robust second phase in addition involved reverse operation of the Na+–Ca2+ exchanger and mitochondrial Ca2+ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na+ current and [Ca2+]i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with INa,P. Describing the magnitude, temporal pattern and sources of Ca2+ increase induced by INa,P may provide novel targets for antiepileptic drug therapy. 相似文献
20.
Ranjan K. Pradhan 《Biophysical journal》2010,98(2):218-230
Sodium-calcium antiporter is the primary efflux pathway for Ca2+ in respiring mitochondria, and hence plays an important role in mitochondrial Ca2+ homeostasis. Although experimental data on the kinetics of Na+-Ca2+ antiporter are available, the structure and composition of its functional unit and kinetic mechanisms associated with the Na+-Ca2+ exchange (including the stoichiometry) remains unclear. To gain a quantitative understanding of mitochondrial Ca2+ homeostasis, a biophysical model of Na+-Ca2+ antiporter is introduced that is thermodynamically balanced and satisfactorily describes a number of independent data sets under a variety of experimental conditions. The model is based on a multistate catalytic binding mechanism for carrier-mediated facilitated transport and Eyring's free energy barrier theory for interconversion and electrodiffusion. The model predicts the activating effect of membrane potential on the antiporter function for a 3Na+:1Ca2+ electrogenic exchange as well as the inhibitory effects of both high and low pH seen experimentally. The model is useful for further development of mechanistic integrated models of mitochondrial Ca2+ handling and bioenergetics to understand the mechanisms by which Ca2+ plays a role in mitochondrial signaling pathways and energy metabolism. 相似文献