Nonuniform distribution of Ca(2+) uptake sites within human neutrophils

The rate at which Ca(2+) returns towards the basal concentration is controlled by the action of Ca(2+) pumps, both on the plasma membrane and on organelles within the cytosol. The distribution of Ca(2+) uptake sites within the cytosol was investigated using rapid confocal imaging (55 ms/frame) of fluo3-loaded human neutrophils. In some zones within the cell, the uptake of Ca(2+) from the cytosol followed a single exponential time course, whereas in others, there was accelerated kinetics after about 3 s. Using the full array of data, to produce a cell-map of Ca(2+) uptake rates a clear nonuniformity of Ca(2+) uptake sites throughout the neutrophil cytosol was observed. The location of the Ca(2+) uptake sites did not correlate with the granules or the main body of the nucleus, but Ca(2+) uptake was highest near the vestigial Golgi/ER, the edges of the nuclear lobes and at the leading cell edge. The possibility exists that the nonuniform distribution of Ca(2+) uptake sites plays a role in restricting Ca(2+) signals with the neutrophil cytosol.     

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system

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 ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).     

Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2

Clusters of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), with physiological sizes of approximately six CD59 molecules, recruit Galphai2 and Lyn via protein-protein and raft interactions. Lyn is activated probably by the Galphai2 binding in the same CD59 cluster, inducing the CD59 cluster's binding to F-actin, resulting in its immobilization, termed stimulation-induced temporary arrest of lateral diffusion (STALL; with a 0.57-s lifetime, occurring approximately every 2 s). Simultaneous single-molecule tracking of GFP-PLCgamma2 and CD59 clusters revealed that PLCgamma2 molecules are transiently (median = 0.25 s) recruited from the cytoplasm exclusively at the CD59 clusters undergoing STALL, producing the IP(3)-Ca(2+) signal. Therefore, we propose that the CD59 cluster in STALL may be a key, albeit transient, platform for transducing the extracellular GPI-AR signal to the intracellular IP(3)-Ca(2+) signal, via PLCgamma2 recruitment. The prolonged, analogue, bulk IP(3)-Ca(2+) signal, which lasts for more than several minutes, is likely generated by the sum of the short-lived, digital-like IP(3) bursts, each created by the transient recruitment of PLCgamma2 molecules to STALLed CD59.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) 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 Ca(2+) release in heart cells under various conditions.     

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。     

Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

The Na(+)-Ca(2+) exchanger (NCX) mediated Ca(2+) fluxes are essential for handling Ca(2+) homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca(2+) binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca(2+) binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of "occluded" Ca(2+). Here we investigate the effects of CBD2 variants on Ca(2+) occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca(2+) occlusion, whereas after dissociation of the first Ca(2+) ion the second Ca(2+) ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca(2+) exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca(2+) dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.     

Symmetry properties of the Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles

The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.     

Intracellular Ca2+ oscillations, a potential pacemaking mechanism in early embryonic heart cells

Early (E9.5-E11.5) embryonic heart cells beat spontaneously, even though the adult pacemaking mechanisms are not yet fully established. Here we show that in isolated murine early embryonic cardiomyocytes periodic oscillations of cytosolic Ca(2+) occur and that these induce contractions. The Ca(2+) oscillations originate from the sarcoplasmic reticulum and are dependent on the IP(3) and the ryanodine receptor. The Ca(2+) oscillations activate the Na(+)-Ca(2+) exchanger, giving rise to subthreshold depolarizations of the membrane potential and/or action potentials. Although early embryonic heart cells are voltage-independent Ca(2+) oscillators, the generation of action potentials provides synchronization of the electrical and mechanical signals. Thus, Ca(2+) oscillations pace early embryonic heart cells and the ensuing activation of the Na(+)-Ca(2+) exchanger evokes small membrane depolarizations or action potentials.     

Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca(2+)-selective dye Oregon Green 488 BAPTA-1 (OG, 100 microM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca(2+) entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca(2+)]i at individual hotspots rose with a time constant tau1 approximately 70 ms and decayed with a bi-exponential time-course (tau2 approximately 160, tau3 approximately 2500 ms) following a 160 ms depolarization to -20 mV. With repeated stimulation [Ca(2+)]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca(2+) channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca(2+)channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1s), Ca(2+) was able to reach the cell apical ciliated pole. The effective Ca(2+) diffusion constant, measured from the progression of Ca(2+) wavefronts in the cytosol, was approximately 57 microm(2)/s. Our results indicate that in these hair cells, buffered diffusion of Ca(2+) proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers.     

Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5 degrees, 15 degrees, and 25 degrees C. The two sites in a molecule were equivalent on Mg2(+)-Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25 degrees C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin on Ca2+ and Mg2+ binding and Mg2(+)-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236-2240]. Although no major conformational changes were noted between Ca2(+)- and Mg2(+)-bound forms of toad parvalbumin, the conformational difference was larger in Ca2+ (or Mg2+) binding to site 1 than site 2. This may indicate that the metal-free form is much less stable than any form with Ca2+ (or Mg2+) bound at one site at least. On Mg2(+)-Ca2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former indicates the sequestering of nonpolar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2(+)-Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2(+)-Ca2+ exchange.     

The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).     

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.     

The permeability transition pore as a Ca(2+) release channel: new answers to an old question

Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals.     

A common Ca2+-driven interdomain module governs eukaryotic NCX regulation

Na(+)/Ca(2+) exchanger (NCX) proteins mediate Ca(2+)-fluxes across the cell membrane to maintain Ca(2+) homeostasis in many cell types. Eukaryotic NCX contains Ca(2+)-binding regulatory domains, CBD1 and CBD2. Ca(2+) binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca(2+). To further elucidate the underlying regulatory mechanisms, we determined the 2.7 ? crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca(2+) binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca(2+)-driven interdomain switch controls slow dissociation of "occluded" Ca(2+) from the primary sensor and thus dictates Ca(2+) sensing dynamics. In the Ca(2+)-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca(2+)-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.