Daniel Khananshvili

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange

Mitochondrial Ca2+ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na+-dependent mechanism mediates mitochondrial Ca2+ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na+/Ca2+ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca2+ and Na+ fluorescent imaging, we demonstrate that mitochondrial Na+-dependent Ca2+ efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca2+ transport was inhibited, moreover, by CGP-37157 and exhibited Li+ dependence, both hallmarks of mitochondrial Na+-dependent Ca2+ efflux. Finally, NCLX-mediated mitochondrial Ca2+ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na+/Ca2+ exchanger.

Sodium-calcium exchangers (NCX): molecular hallmarks underlying the tissue-specific and systemic functions

NCX proteins explore the electrochemical gradient of Na+ to mediate Ca2+-fluxes in exchange with Na+ either in the Ca2+-efflux (forward) or Ca2+-influx (reverse) mode, whereas the directionality depends on ionic concentrations and membrane potential. Mammalian NCX variants (NCX1-3) and their splice variants are expressed in a tissue-specific manner to modulate the heartbeat rate and contractile force, the brain’s long-term potentiation and learning, blood pressure, renal Ca2+ reabsorption, the immune response, neurotransmitter and insulin secretion, apoptosis and proliferation, mitochondrial bioenergetics, etc. Although the forward mode of NCX represents a major physiological module, a transient reversal of NCX may contribute to EC-coupling, vascular constriction, and synaptic transmission. Notably, the reverse mode of NCX becomes predominant in pathological settings. Since the expression levels of NCX variants are disease-related, the selective pharmacological targeting of tissue-specific NCX variants could be beneficial, thereby representing a challenge. Recent structural and biophysical studies revealed a common module for decoding the Ca2+-induced allosteric signal in eukaryotic NCX variants, although the phenotype variances in response to regulatory Ca2+ remain unclear. The breakthrough discovery of the archaebacterial NCX structure may serve as a template for eukaryotic NCX, although the turnover rates of the transport cycle may differ ∼103-fold among NCX variants to fulfill the physiological demands for the Ca2+ flux rates. Further elucidation of ion-transport and regulatory mechanisms may lead to selective pharmacological targeting of NCX variants under disease conditions.

NCLX: the mitochondrial sodium calcium exchanger.

The free Ca(2+) concentration within the mitochondrial matrix ([Ca(2+)]m) regulates the rate of ATP production and other [Ca(2+)]m sensitive processes. It is set by the balance between total Ca(2+) influx (through the mitochondrial Ca(2+) uniporter (MCU) and any other influx pathways) and the total Ca(2+) efflux (by the mitochondrial Na(+)/Ca(2+) exchanger and any other efflux pathways). Here we review and analyze the experimental evidence reported over the past 40years which suggest that in the heart and many other mammalian tissues a putative Na(+)/Ca(2+) exchanger is the major pathway for Ca(2+) efflux from the mitochondrial matrix. We discuss those reports with respect to a recent discovery that the protein product of the human FLJ22233 gene mediates such Na(+)/Ca(2+) exchange across the mitochondrial inner membrane. Among its many functional similarities to other Na(+)/Ca(2+) exchanger proteins is a unique feature: it efficiently mediates Li(+)/Ca(2+) exchange (as well as Na(+)/Ca(2+) exchange) and was therefore named NCLX. The discovery of NCLX provides both the identity of a novel protein and new molecular means of studying various unresolved quantitative aspects of mitochondrial Ca(2+) movement out of the matrix. Quantitative and qualitative features of NCLX are discussed as is the controversy regarding the stoichiometry of the NCLX Na(+)/Ca(2+) exchange, the electrogenicity of NCLX, the [Na(+)]i dependency of NCLX and the magnitude of NCLX Ca(2+) efflux. Metabolic features attributable to NCLX and the physiological implication of the Ca(2+) efflux rate via NCLX during systole and diastole are also briefly discussed.

Kinetic and equilibrium properties of regulatory calcium sensors of NCX1 protein.

The crystal structures of the CBD1 and CBD2 domains of the Na+/Ca2+ exchanger protein (NCX1) provided a major breakthrough in Ca2+-dependent regulation of NCX1, although the dynamic aspects of the underlying molecular mechanisms are still not clear. Here we provide new experimental approaches for evaluating the kinetic and equilibrium properties of Ca2+ interaction with regulatory sites by using purified preparations of CBD1, CBD2, and CBD12 proteins. CBD12 binds ∼6 Ca2+ ions (mol/mol), whereas the binding of only ∼2 Ca2+ ions is observed (with a Hill coefficient of nH = ∼2) either for CBD1 or CBD2. In the absence of Mg2+, CBD1 has a much higher affinity for Ca2+ (Kd = 0.3 ± 1.2 μm) than CBD2 (Kd = 5.0 ± 1.2 μm). The Ca2+ dissociation from CBD2 (koff = 230 ± 70 s–1) is at least 25 times faster than from CBD1 (koff = 10 ± 3 s–1), whereas the kon values indicate fast kinetics for Ca2+ binding (kon = koff/Kd = 107–108 m–1 s–1) for both CBDs. At 2–5 mm Mg2+, both CBDs bind Ca2+, with a Kd of 1–2 μm (Mg2+ has very little effect on Ca2+ off rates). Mg2+ cannot occupy the primary site of CBD2, whereas the other Ca2+ sites of CBDs interact with Mg2+ as well. There is no competition between Na+ and Ca2+ for any CBD site. The kinetically diverse Ca2+ sensors may sense differentially the dynamic swings in [Ca2+] within specific subcellular compartments (dyadic cleft, submembrane space, bulk cytosol, etc.).

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca2+-binding sites contribute to NCX regulation. Two of them are located on CBD1 (Kd = ∼0.2 μm), and one is on CBD2 (Kd = ∼5 μm). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable Kd values but differ dramatically in their Ca2+ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca2+ ions (kr = 280 s−1, kf = 7 s−1, and ks = 0.4 s−1), whereas the dissociation of two Ca2+ ions from CBD1 (kf = 16 s−1) and one Ca2+ ion from CBD2 (kr = 125 s−1) is monophasic. Insertion of seven alanines into the linker (CBD12–7Ala) abolishes slow dissociation of Ca2+, whereas the kinetic and equilibrium properties of three Ca2+ sites of CBD12–7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca2+ on/off kinetics at a specific CBD1 site by 50–80-fold, thereby representing Ca2+ “occlusion” at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are “linker-independent,” so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.

Proton-sensing Ca2+ Binding Domains Regulate the Cardiac Na+/Ca2+ Exchanger

The cardiac Na+/Ca2+ exchanger (NCX) regulates cellular [Ca2+]i and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C2 regulatory domains, Ca2+ binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, INCX, whereas [H+]i was varied using an ammonium chloride “rebound” method at constant extracellular pH 7.4. At pHi = 7.2 and [Ca2+]i < 120 nm, INCX was less than 4% that of its maximally Ca2+-activated value. INCX increases steeply at [Ca2+]i between 130–150 nm with a Hill coefficient (nH) of 8.0 ± 0.7 and K0.5 = 310 ± 5 nm. At pHi = 6.87, the threshold of Ca2+-dependent activation of INCX was shifted to much higher [Ca2+]i (600–700 nm), and the relationship was similarly steep (nH = 8.0±0.8) with K0.5 = 1042 ± 15 nm. The Vmax of Ca2+-dependent activation of INCX was not significantly altered by low pHi. The Ca2+ affinities for CBD1 (0.39 ± 0.06 μm) and CBD2 (Kd = 18.4 ± 6 μm) were exquisitely sensitive to [H+], decreasing 1.3–2.3-fold as pHi decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C2 regulatory domains CBD1 and CBD2.

The peptide ”FRCRCFa”, dialysed intracellularly, inhibits the Na/Ca exchange in rabbit ventricular myocytes with high affinity

Abstract We investigated the effect in rabbit ventricular myocytes of ”FRCRCFa”, a newly developed peptide inhibitor of the Na/Ca exchange. Myocytes were whole-cell patch clamped and experiments were carried out at 36°C. The Na/Ca exchange was measured selectively, by blocking interfering ion channel currents and the Na/K pump, as the membrane current which could be inhibited by 5 mM nickel (Ni; a known blocker of the Na/Ca exchange). Increasing concentrations of FRCRCFa dialysed into the cell from the patch-pipette inhibited the Na/Ca exchange current. The dose/response curve could be fitted by a function for co-operative ligand binding, which predicted a KD for FRCRCFa-mediated inhibition of 22.7 ± 3.7 nM, with a Hill coefficient of 0.61 ± 0.06. Pipette FRCRCFa concentrations of 1 μM and above were sufficient to cause complete inhibition of Na/Ca exchange current. The inhibitory effect of FRCRCFa was independent of membrane potential and relatively selective: 10 μM FRCRCFa dialysed into the cell had no effect on the L-type Ca current and delayed rectifier and inward rectifier K currents. Thus FRCRCFa appears to be a potent and relatively selective inhibitor of the Na/Ca exchange in intact cardiac myocytes, and may be of value for studies of the Na/Ca exchange.

[53] Selective extraction and reconstitution of F1 subunits from Rhodospirillum rubrum chromatophores

Publisher Summary his chapter describes the selective extraction and reconstitution of F 1 subunits from Rhodospirillurn rubrum chromatophores. The F 1 -ATPase isolated from membranes of mitochondria, bacteria, and chloroplasts is very similar, containing five nonidentical polypeptide subunits: α, β, γ, δ, and ɛ. Using R. rubrum chromatophores, two F 1 subunits—β and γ—are extracted in two consecutive steps, leaving all other F1 subunits attached to the chromatophore membrane. The procedures developed for the extractions, for purification of the isolated subunits, and for their reconstitution into the depleted chromatophores are described. The isolated β and γ subunits have no activity by themselves, although their extraction leads to complete loss of photophosphorylation and ATPase activities of the depleted chromatophores. The subunits are therefore identified during the extraction and purification procedures by their reconstitutive activity. This is their capacity to rebind to depleted chromatophores and restore their lost activities. The experimental system for measuring the reconstitutive activity involves two steps: (1) reconstitution of the isolated subunits into the depleted chromatophores, and (2) assay of the reconstituted chromatophores for restored activities.

SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells

Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, responsible for cardiac automaticity. Although the cardiac pacemaker was discovered more than a hundred years ago, the pacemaker mechanisms remain controversial. We used human embryonic stem cell-derived cardiomyocytes to study the embryonic cardiac automaticity of the human heart. We identified a previously unrecognized Ca2+-activated K+ channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca2+-activated K+ channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers. Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the “voltage-clock,” where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the “Ca2+ clock,” where cyclical release of Ca2+ from Ca2+ stores depolarizes the membrane during diastole via activation of the Na+–Ca2+ exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called “voltage and Ca2+ clock” pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the “voltage or Ca2+ clock” produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca2+-activated intermediate K+ conductance (IKCa, KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IKCa inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IKCa appears to play a crucial role in human embryonic cardiac automaticity.

The mitochondrial Na+/Ca2+ exchanger upregulates glucose dependent Ca2+ signalling linked to insulin secretion.

Mitochondria mediate dual metabolic and Ca2+ shuttling activities. While the former is required for Ca2+ signalling linked to insulin secretion, the role of the latter in β cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca2+ transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na+/Ca2+ exchanger that is linked to Ca2+ signalling in MIN6 and primary β cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX) or of its activity, by a dominant negative construct (dnNCLX), enhanced mitochondrial Ca2+ influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca2+. Importantly, NCLX controlled the rate and amplitude of cytosolic Ca2+ changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca2+ signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na+/Ca2+ exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca2+ signals thereby regulating the temporal pattern of insulin secretion in β cells.