Papers by D. Khananshvili
Proceedings of the National Academy of Sciences, 2009
Mitochondrial Ca 2+ efflux is linked to numerous cellular activities and pathophysiological proce... more Mitochondrial Ca 2+ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na + -dependent mechanism mediates mitochondrial Ca 2+ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na + /Ca 2+ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca 2+ and Na + fluorescent imaging, we demonstrate that mitochondrial Na + -dependent Ca 2+ 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 Ca 2+ transport was inhibited, moreover, by CGP-37157 and exhibited Li + dependence, both hallmarks of mitochondrial Na + -dependent Ca 2+ efflux. Finally, NCLX-mediated mitochondrial Ca 2+ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the...
Journal of Biological Chemistry, Jan 5, 1993
The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides... more The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides inhibit Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles. FMRFa itself has a low inhibitory potency (IC50 = 750 microM) which completely resides in its COOH-terminal RFa portion. The physiologically active analog FLRFa is 10-fold more potent (IC50 = 60 microM). Two other substitutions of the Met2 in FMRFa, by either Ile or Lys increase inhibitory potency 7- and 50-fold, respectively. The inhibitory potency increases 300-500-fold if the NH2-terminal Phe1 in FMRFa is substituted by either Val or His (IC50 = 1-2 microM). The inhibitory activity of WnLRFa (IC50 = 40 microM) is lost when either the NH2-terminal amino group is acylated or the NH2-terminal Trp1 is deleted. These data suggest that the COOH-terminal portion is essential for the basic low potency inhibition of Na(+)-Ca2+ exchange, whereas the NH2-terminal portion is important for the potentiation of the inhibitory activity...
Abstract. Various studies on humans and experimental mammals showed that d-sotalol and tedisamil ... more Abstract. Various studies on humans and experimental mammals showed that d-sotalol and tedisamil (class III antiarrhythmic drugs with positive inotropic effect) facilitate spontaneous ventricular defibrillation Following our previous results, we summarized that spontaneous ventricular defibrillation requires high level of intercellular coupling and synchronization, both of which depends on intracellular free Ca 2+ concentration We hypothesized that any antiarrhythmic compound that facilitates spontaneous defibrillation, including d-sotalol and tedisamil, should prevent intracellular free Ca 2+ overload most likely by elevating cAMP level and enhancing cAMP -related Ca 2+ uptake of the sarcoplasmic reticulum (SR) The aim of the present study was to examine the role of the SR uptake function in their effect against Ca 2+ overload Methods The effect of d-sotalol, tedisamil and dbcAMP on increased intracellular Ca 2+ level were examined in cultured rat cardiomyocytes during blockade of ...
Journal of Biological Chemistry, Oct 10, 1982
We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed c... more We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed completely the beta-subunit of their coupling factor ATPase complex leaving the other four subunits attached to the membrane (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752). Further treatment of these beta-less chromatophores with LiBr, under the described optimal conditions, resulted in specific removal of one additional subunit, the gamma-subunit, and both subunits were purified to homogeneity. The beta, gamma-less chromatophores as well as the beta-less ones lost their ATP-linked activities, but retained their light-induced proton uptake, resulting in the formation of an electrochemical gradient of protons composed of both a pH gradient and a membrane potential. These results indicate that the removed beta and gamma subunits cannot be an integral part of an H+ gate in the R. rubrum chromatophore membrane. Each of the removed subunits could bind ...
Biochemical and biophysical research communications, 1982
Biochemistry, 1988
Phosphorylation by ATP of E x *Ca/sub 2/ (sarcoplasmic reticulum vesicles (SRV) with bound /sup 4... more Phosphorylation by ATP of E x *Ca/sub 2/ (sarcoplasmic reticulum vesicles (SRV) with bound /sup 45/Ca/sup 2 +/) during 5-10 ms leads to the occlusion of 2 *Ca/sup 2 +/EP/sub tot/ (quench by ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone) in both empty (10 ..mu..M free Ca/sup 2 +//sub in/) or loaded SRV (20-40 mM free Ca/sup 2 +//sub in/). The rate of Ca/sup 2 +/ internalization from the occluded E approx. P x *Ca/sub 2/ was measured by using ADP + EGTA quench; a Ca/sup 2 +/ ion that is not removed by this quench is defined as internalized. These data show that the two Ca/sup 2 +/ ions are internalized sequentially, presumably from separate sequential sites in the channel. (/sup 32/P)EP x Ca/sub 2/ obtained by rapid mixing of E x Ca/sub 2/ with (..gamma..-/sup 32/P)ATP and EGTA disappears in a biphasic time course with a lag corresponding to approx. 34 s/sup -1/, followed by EP* decay with a rate constant of approx. 17 s/sup -1/. This s...
Binding sites foronePiandtwoATPorADP molecules havebeenidentified ontheisolated, reconstitutively... more Binding sites foronePiandtwoATPorADP molecules havebeenidentified ontheisolated, reconstitutively active .3subunit fromtheRhodospirillum rubrumFo-F1 ATP synthase. Chemical modification ofthis.8subunit bythe histidine reagent diethyl pyrocarbonate orbythecarboxyl groupreagent Woodword's reagent K results incomplete inhibition ofPibinding to(3.Thesamereagents inhibit the binding ofATPtoaMg-dependent low-affinity site butnotto aMg-independent high-affinity site onthis13subunit. The binding stoichiometry ofADPtoeither site isnotaffected by these modifications. The/3subunit modified byeither oneof thesereagents retains itscapacity torebindtofl-less chromatophores butnotitsability torestore their photo- phosphorylation. Theseresults indicate that thelow-affinity Pi binding site on.3islocated atthebinding site ofthey- phosphate groupofATP intheMg-dependent low-afflinity nucleotide binding site. Thissitecontains histidine and carboxyl groupresidues, bothofwhicharerequired forthe binding...
Current opinion in cell biology, 1990
Exchange-only systems (otherwise known as antiporters or counter-transport systems) catalyze a co... more Exchange-only systems (otherwise known as antiporters or counter-transport systems) catalyze a coupled, stoichiometric exchange of two substances located at opposite sides of the membrane (Stein, Tran.port and D$ @ion Across Cell Membrane, Academic Press, 1986). Antiporters are carrier systems, exposing ion binding sites at the opposite sides of the membrane during dilferent stages of the catalytic turnover. This machinery is different from the channel mechanisms, in which the cation translocating device may exist in two main states, open or closed (Iauger, Physiol Rev 1987,67:1296-1331), but, when open, with simultaneous access to the channel interior from both sides of the membrane.
UNLABELLED Various studies on humans and experimental mammals showed that d-sotalol and tedisamil... more UNLABELLED Various studies on humans and experimental mammals showed that d-sotalol and tedisamil (class III antiarrhythmic drugs with positive inotropic effect) facilitate spontaneous ventricular defibrillation. Following our previous results, we summarized that spontaneous ventricular defibrillation requires high level of intercellular coupling and synchronization, both of which depends on intracellular free Ca2+ concentration. We hypothesized that any antiarrhythmic compound that facilitates spontaneous defibrillation, including d-sotalol and tedisamil, should prevent intracellular free Ca2+ overload most likely by elevating cAMP level and enhancing cAMP-related Ca2+ uptake of the sarcoplasmic reticulum (SR). The aim of the present study was to examine the role of the SR uptake function in their effect against Ca2+ overload. METHODS The effect of d-sotalol, tedisamil and dbcAMP on increased intracellular Ca2+ level were examined in cultured rat cardiomyocytes during blockade of S...
Biochemistry, 1988
Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during... more Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone] in both "empty" (10 microM free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E approximately P.*Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot is internalized from 45Ca-labeled E approximately P.*Ca2 with a first-order rate constant of kl = 34 s-1. Empty SRV take up 2 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 approximately 1.3 mM and a Hill coefficient ...
Life Sciences, 1992
Opiate agonists and antagonists inhibit Na(+)-Ca2+ exchange in the isolated cardiac sarcolemma ve... more Opiate agonists and antagonists inhibit Na(+)-Ca2+ exchange in the isolated cardiac sarcolemma vesicles. Non-opioid stereoisomers (dextrorphan, Mr 1542MS, WIN 44,441-3) display effects similar to their opioid isomers (levorphanol, Mr 1543MS, WIN 44,441-2) suggesting that inhibition is not mediated by opiate receptors. Naloxone (permeable) and methylnaloxone (impermeable) inhibit the Na(+)-Ca2+ exchange similarly, suggesting an extravesicular location of inhibitory site. The inhibitory potency of naloxone is pH-independent in the range of 7.4-9.1, suggesting that the charge-carrying properties of drug-protein interactions are not altered under the tested conditions. Opiates display similar dose-response relationships for Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The opiate-induced inhibition is complete and noncompetitive in regard to extravesicular calcium. These data suggest that opiates do not bind to the Ca(2+)-binding domain (A-site), but they may interest either with the Na(+)-binding site (B-site) or with a putative opiate-binding site, presumably located outside of the ion-binding vicinity. Further studies on structure-activity relationship might lead to the discovery of potent and more specific inhibitors of cardiac Na(+)-Ca2+ exchanger. A possible relevance of these findings to some non-opioid pharmacological effects of naloxone on the cardiac muscle is suggested.
Biochemistry, 1990
In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in th... more In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.
Biochemical and Biophysical Research Communications, 1981
ABSTRACT
The Journal of biological chemistry, Jan 10, 1982
We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed c... more We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed completely the beta-subunit of their coupling factor ATPase complex leaving the other four subunits attached to the membrane (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752). Further treatment of these beta-less chromatophores with LiBr, under the described optimal conditions, resulted in specific removal of one additional subunit, the gamma-subunit, and both subunits were purified to homogeneity. The beta, gamma-less chromatophores as well as the beta-less ones lost their ATP-linked activities, but retained their light-induced proton uptake, resulting in the formation of an electrochemical gradient of protons composed of both a pH gradient and a membrane potential. These results indicate that the removed beta and gamma subunits cannot be an integral part of an H+ gate in the R. rubrum chromatophore membrane. Each of the removed subunits could bind ...
The Journal of biological chemistry, Jan 15, 1985
Photophosphorylation and ATPase activities were restored to beta-less Rhodospirillum rubrum chrom... more Photophosphorylation and ATPase activities were restored to beta-less Rhodospirillum rubrum chromatophores by their reconstitution with purified beta-subunits of either R. rubrum F1-ATPase (Rr beta) or Escherichia coli F1-ATPase (Ec beta). In the homologous reconstituted system both activities were restored to the same extent, whereas in the hybrid system ATP synthesis was restored to about 10% when the hydrolysis was restored to 200%. This difference in rates of synthesis and hydrolysis was not due to any general uncoupling effect of Ec beta leading to an increased membrane permeability to protons, because with both hybrid and homologous systems an identical light-induced quenching of quinacrine fluorescence was observed. They differed, however, in ATP-driven quenching of quinacrine fluorescence, which was much lower in the hybrid system. These results suggest that the hybrid has a decreased capacity for proton-translocation through the membrane-bound Fo channel during ATP hydrolys...
In multidomain proteins, interdomain linkers allow an efficient transfer of regulatory informatio... more In multidomain proteins, interdomain linkers allow an efficient transfer of regulatory information, although it is unclear how the information encoded in the linker structure coins dynamic coupling. Allosteric regulation of NCX proteins involves Ca 2+ -driven tethering of regulatory CBD1 and CBD2 (through a salt bridge network) accompanied by alignment of CBDs and Ca 2+ occlusion at the interface of the two CBDs. Here we investigated "alanine-walk" substitutions in the CBD1− CBD2 linker (501-HAGIFT-506) and found that among all linker residues, only G503 is obligatory for Ca 2+ -induced reorientations of CBDs and slow dissociation of occluded Ca 2+ . Moreover, swapping between positions A502 and G503 in the CBD1−CBD2 linker results in a complete loss of slow dissociation of occluded Ca 2+ , meaning that dynamic coupling of CBDs requires an exact pose of glycine at position 503. Therefore, accumulating data revealed that position 503 occupied by glycine is absolutely required for Ca 2+ -driven tethering of CBDs, which in turn limits the linker's flexibility and, thus, restricts CBD movements. Because G503 is extremely well conserved in eukaryotic NCX proteins, the information encoded in G503 is essential for dynamic coupling of the two-domain CBD tandem and, thus, for propagation of the allosteric signal.
Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology... more 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 I f causes diastolic depolarization that triggers action potential cycling; and (ii) the " Ca 2+ clock, " where cyclical release of Ca 2+ from Ca 2+ stores depolarizes the membrane during diastole via activation of the Na + –Ca 2+ 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 Ca 2+ 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 Ca 2+ clock " produced a similar depo-larization 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 Ca 2+-activated intermediate K + conduc-tance (IK Ca , KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK Ca inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK Ca appears to play a crucial role in human embryonic cardiac automaticity. Ca2 +-activated K + channel SK4 | voltage clock | calcium clock | Na +-Ca 2+ exchanger | hyperpolarization-activated cyclic nucleotide-gated channel W hereas in early embryonic stages all cardiomyocytes are initially endowed with pacemaker activity, during heart development , most cardiac cells will differentiate into working myo-cardium lacking pacemaker properties. Only a small population of embryonic cardiomyocytes will form the sinoatrial node (SAN), the atrioventricular node, and the bundle of His (1). A crucial requirement for rhythmic automaticity is the existence of inward currents at diastolic potentials and a subtle dynamic integration of sarcolemmal ion channels, transporters, and Ca 2+ cycling proteins (2). Various ionic currents finely orchestrate rhythmic automaticity and are referred to as a voltage clock, including the pacemaker or funny current (I f), L-type, and T-type Ca 2+ currents (3–10). A Ca 2+-dependent pacemaker mechanism referred to as a Ca 2+ clock was also suggested to be a major player for automa-ticity, where the rhythmic local Ca 2+ release from the sarcoplasmic reticulum (SR) drives SAN pacemaker activity. SR Ca 2+ release via ryanodine receptors (RyRs) is thought to activate the forward mode of the electrogenic sarcolemmal Na + –Ca 2+ exchanger (NCX), which generates an inward current contributing to the late diastolic depolarization (DD), before the next action potential (AP) (7, 11). However, the cardiac pacemaker mechanisms remain unclear and controversial (12–15). Embryonic stem cells differentiate in vitro into spontaneously beating multicellular cardiomyocyte clusters within embryoid bodies (EBs), while recapitulating developmental stages of em-bryonic cardiomyogenesis (16–27). Thus, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) may provide insights into the pacemaker mechanisms of embryonic cardiac development. In this work, we used the current-and voltage-clamp configurations of the whole-cell patch-clamp technique to record, in the same beating hESC-CM, basal automaticity and ionic currents. This analysis revealed three main pacemaker phenotypes. The first was highly sensitive to I f current inhibition and insensitive to NCX blockade (28–30). The second cell population exhibited a pacemaker phenotype insensitive to I f current inhibition, but highly responsive to NCX blockers. The third hESC-CM population displayed pacemaker features that were sensitive to both I f and NCX blockers, indicating that voltage and Ca 2+ dependent pacemaker mechanisms can coexist in the same cell. Following exposure to blockers, all three pacemaker phenotypes shared a depolarizing drift of the maximal diastolic potential (MDP) that culminated by cessation of APs, suggesting that they converge to a common pacemaker component, which we identified by patch-clamp recording, real-time PCR, Western blotting, and immunocy-tochemistry as belonging to the intermediate-conductance Ca 2+-activated K + channels (IK Ca , KCa3.1 or SK4). Remarkably, IK Ca blockers (31, 32) inhibited the pacemaker in beating hESC-CMs, thereby leading to bradycardia, MDP depolarization, and ultimate suppression of automaticity. The pacemaker activity was sensitive 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 Ca 2+-activated K + channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca 2+-activated K + channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers.
The Journal of biological chemistry, Jan 25, 1991
The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separa... more The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values range...
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Papers by D. Khananshvili