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1.
Ca2+-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin
light chain, or calmodulin as the Ca2+ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca2+ signals into allosteric conformational changes of contractile proteins. While these Ca2+ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The
Ca2+ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting
of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca2+ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes
encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin
I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific
adapter to couple the Ca2+ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two
structure–function domains: the Ca2+ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca2+ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged
to constitute the Ca2+ signal transduction pathways governing nonmuscle cell motility.
Received: 15 April 1999 / Accepted: 15 July 1999 相似文献
2.
Parallel measurements of Ca2+ uptake, oxygen consumption, endogenous Mg2+ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca2+ in the presence of phosphate than rat liver mitochondria. The extent of Mg2+ efflux and swelling induced by Ca2+ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg2+ as a consequence of Ca2+ recycling across the membrane as induced by phosphate. Exogenous Mg2+ protects liver mitochondria against the deleterious effects of Ca2+ by inhibiting a ruthenium red-insensitive Ca2+ efflux induced by phosphate. Phosphate does not induce recycling of Ca2+ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca2+ recycling. In heart mitochondria the recycling of Ca2+ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca2+ is subject to metabolic control. In addition it has been observed that Sr2+does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca2+ than the ruthenium red-sensitive influx transporter. 相似文献
3.
Ca2+ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca2+ binding in the NH2-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca2+ slowed HDX up to 2 orders of magnitude within the NH2-lobe and the NH2-lobe-associated TnI switch helix, implying that Ca2+ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca2+ and actin. Ca2+-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca2+ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca2+-mediated regulation: the coiled-coil''s NH2-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca2+-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca2+, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca2+ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features. 相似文献
4.
The interaction of troponin molecules on the thin filament with Ca2+ plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca2+ and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca2+ concentration using fluo-3 AM in the same frog skeletal muscle during twitch elicited by a single electrical pulse at 16°C. In an overstretched muscle, the intensity of the meridional reflection from troponin at 1/38.5 nm−1 began to change at 4 ms after the stimulus, reached a peak at 10 ms, and returned to the resting level with a halftime of 25 ms. The concentration of troponin-bound Ca2+ began to increase at 1-2 ms after the stimulus, reached a peak at 5 ms, and returned to the resting level with a halftime of 40 ms, indicating that troponin begins to change conformation only after a sizable amount of Ca2+ has bound to it, and returns to the resting structure even when there is still some bound Ca2+. In a muscle with a filament overlap, crossbridge formation appears to slow down Ca2+ release from troponin and have a large effect on its conformation. 相似文献
5.
Akira Kawashima Fumie Shiraishi Iwao Ohtsuki Kazuhiko Yamamoto 《Molecular and cellular biochemistry》1994,132(2):173-177
In order to compare the role of the Ca2+-receptive protein (troponin), in the characteristic myofibrillar contractile response of chicken fast and slow skeletal muscles, the troponin in both kinds of myofibrils were partially exchanged, under slightly acidic conditions. The Ca2+- or Sr2+-activation of the ATPase of fast (or slow) skeletal myofibrils hybridized with slow (or fast) skeletal troponin profiles were also investigated. The results indicated that the Ca2+- or Sr2+-affinity of the myofibrillar ATPase activity were related to the species of troponin. This procedure for replacing troponin in myofibrils under physiological conditions in thus considered to be useful for the study of the Ca2+-regulatory mechanism in myofibrillar contraction. 相似文献
6.
The effect of Sr2+ on the set point for external Ca2+ was studied in rat heart and liver mitochondria with the aid of a Ca2+-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca2+ influx on the Ca2+ uniporter and efflux by various mechanisms. We studied the Ca2+-Na+ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr2+ was found to shift the set points towards lower external Ca2+ both in heart mitochondria under conditions of Ca2+-Na+ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca2+ efflux by Sr2+ taken up by the mitochondria, while Sr2+ efflux was too slow to be measurable. 相似文献
7.
A variety of chemically different prooxidants causes Ca2+ release from mitochondria. The prooxidant-induced Ca2+ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein ADP-ribosylation. When the released Ca2+ is excessively cycled by mitochondria they are damaged. This leads to uncoupling, a decreased ATP supply, and a decreased ability of mitochondria to retain Ca2+. Excessive Ca2+ cycling by mitochondria will deprive cells of ATP. As a result, Ca2+ ATPases of the endoplasmic (sarcoplasmic) reticulum and the plasma membrane are stopped. The rising cytosolic Ca2+ level cannot be counterbalanced due to damage of mitochondria which, under normoxic conditions, act as safety device against increased cytosolic Ca2+. It is proposed that prooxidants are toxic because they impair the ability of mitochondria to retain Ca2+. 相似文献
8.
Prakorn Chudapongse 《BBA》1976,423(2):196-202
Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+-induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca2+ in the presence of glutamate and inorganic phosphate causes the release of Ca2+ from mitochondria. The phosphoenolpyruvate-stimulated Ca2+ efflux can be observed with mitochondria loaded with low initial Ca2+ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H+ translocation produced by addition of Ca2+ to respiring mitochondria show that phosphoenolpyruvate depresses H+ ejection and enhances H+ uptake by mitochondria. The Ca2+-releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca2+ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca2+ transport. 相似文献
9.
Calcium binding to cardiac troponin C 总被引:1,自引:0,他引:1
The binding of Ca2+ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca2+ concentration by means of a Ca2+-specific electrode. Cardiac troponin C contains three Ca2+-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg2+ which competes with the Ca2+. 相似文献
10.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2008,1784(7-8):1037-1042
Molluscan troponin regulates muscle contraction through a novel Ca2+-dependent activating mechanism associated with Ca2+-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca2+-dependent activation of the Mg-ATPase activity of rabbit-actomyosin–scallop-tropomyosin. In addition, the actin–tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca2+ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin–tropomyosin filaments and is essential for regulation. 相似文献
11.
The ability of mitochondria to capture Ca2+ ions has important functional implications for cells, because mitochondria shape cellular Ca2+ signals by acting as a Ca2+ buffer and respond to Ca2+ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca2+ channel known as the uniporter drives the rapid and massive entry of Ca2+ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca2+ concentrations that are only reached transiently in cells, near Ca2+ release channels. Mitochondria can also take up Ca2+ at low, nanomolar concentrations, but this high affinity mode of Ca2+ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca2+/H+ antiporter that drives the uptake of Ca2+ into mitochondria at nanomolar cytosolic Ca2+ concentrations. In this article, we will review the properties of the Ca2+ import systems of mitochondria and discuss how Ca2+ uptake via an electrogenic 1:1 Ca2+/H+ antiport challenges our current thinking of the mitochondrial Ca2+ uptake mechanism. 相似文献
12.
C S Rossi F D Vasington E Carafoli 《Biochemical and biophysical research communications》1973,50(3):846-852
Ruthenium red, an inhibitor of Ca2+ binding and transport by mitochondria, promotes the release of Ca2+ by mitochondria only if it is added to the assay medium before the accumulation of Ca2+ has been completed. Once essentially all of the Ca2+ in the medium is taken up by mitochondria, ruthenium red does not induce its release. It is proposed that ruthenium red inhibits Ca2+ transport by competing with Ca2+ for Ca2+ binding sites, possibly Ca2+ carrier molecules, on or within the inner mitochondrial membrane. 相似文献
13.
Some features of the Ca2+-transport system in mitochondria of the yeast Endomyces magnusii are considered. The Ca2+ uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca2+ itself. The Na+-independent system responsible for Ca2+ release from Ca2+-preloaded yeast mitochondria was characterized. The rate of the Ca2+ release was proportional to the Ca2+ load, insensitive to cyclosporin A and to Na+, inhibited by La3+, TPP+, Pi, and nigericin, while being activated by spermine. We conclude that Ca2+ release from preloaded E. magnusii yeast mitochondria is mediated by a Na+-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues. A scheme describing an arrangement of the Ca2+ transport system of yeast mitochondria is proposed. 相似文献
14.
Ronald S. Cockrell 《Archives of biochemistry and biophysics》1981,212(2):443-451
The rate, maximum extent of accumulation, and passive release of Ca2+ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca2+ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca2+ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca2+/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca2+ release from mitochondria within the cell (ca. 10 neq Ca2+/min · mg mitochondrial protein for Ca2+ loads up to 800 neq Ca2+/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca2+ accumulation without uncoupling and attenuation of Ca2+ efflux are virtually the same in the cell as in vitro. 相似文献
15.
Muscle contraction is tightly regulated by Ca2+ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca2+ binding site in the NH2 domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D2O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D2O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca2+ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca2+-saturated TnC NH2 domain. More notably, Ca2+ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca2+ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin. 相似文献
16.
Ca2+ stimulates the binding of a variety of prostaglandins (PG) to liver mitochondria. Optimal binding is observed at slightly acidic pH and at concentrations of Ca2+ between 200 and 500 μm. The stimulation of the binding requires the active transport of Ca2+ in mitochondria and is only observed in the absence of permeant anions. The maximal amount of PG bound is about 3 nmol/mg of mitochondrial protein. All PG tested induce efflux of the Ca2+ taken up by mitochondria without impairing the ability of mitochondria to actively accumulate it. Optimal stimulation of the efflux of Ca2+ requires concentrations of PG higher than those used in the PG-binding experiments and is associated with an evident uncoupling of the respiration that follows a Ca2+-induced O2 uptake jump. The “uncoupling” by PG is explained by postulating the entrance of protonated PG into mitochondria, followed by their exit from the organelle as 2:1 complexes with Ca2+. 相似文献
17.
The interaction of La 3+ with mitochondria in relation to respiration-coupled Ca 2+ transport 总被引:9,自引:0,他引:9
La3+ inhibits the respiration-dependent accumulation of Ca2+ by rat liver mitochondria when added in very small amounts (0.1–l.0 nmole per mg protein). However, La3+ itself does not activate respiration. With the use of 140La3+ it was found that La3+ is very rapidly bound to rat liver mitochondria in a respiration-independent process accompanied by loss of H+ to the medium. When both La3+ and Ca2+ are added to mitochondria simultaneously, most of the La3+ but little Ca2+ are bound. La3+ added to mitochondria previously loaded with Ca2+ is tightly bound without discharge of Ca2+. Conversely, when Ca2+ is added to La3+-loaded mitochondria it is not bound nor is the La3+ discharged. La3+ inhibits both high-affinity and low-affinity respiration-independent Ca2+ binding. Isotopic experiments showed that La3+ is, in fact, bound to the same high-affinity sites as Ca2+, in both intact mitochondria and in mitochondrial extracts. It is concluded (1) that La3+ binds to and inhibits the Ca2+ carrier; (2) that La3+ is not transported by the Ca2+ carrier; and (3) that La3+ is, in addition, bound to a large number of external sites on mitochondria for which Ca2+ is not a strong competitor. 相似文献
18.
Ernesto Carafoli 《BBA》2010,1797(6-7):595-606
A number of findings in the 1950s had offered indirect indications that mitochondria could accumulate Ca2+. In 1961, the phenomenon was directly demonstrated using isolated mitochondria: the uptake process was driven by respiratory chain activity or by the hydrolysis of added ATP. It could be accompanied by the simultaneous uptake of inorganic phosphate, in which case precipitates of hydroxyapatite were formed in the matrix, buffering its free Ca2+ concentration. The properties of the uptake process were established in the 1960s and 1970s: the uptake of Ca2+ occurred electrophoretically on a carrier that has not yet been molecularly identified, and was released from mitochondria via a Na+/Ca2+ antiporter. A H+/Ca2+ release exchanger was also found to operate in some mitochondrial types. The permeability transition pore was later also found to mediate the efflux of Ca2+ from mitochondria. In the mitochondrial matrix two TCA cycle dehydrogenases and pyruvate dehydrogenase phosphate phosphatase were found to be regulated in the matrix by the cycling of Ca2+ across the inner membrane. In conditions of cytoplasmic Ca2+ overload mitochondria could store for a time large amounts of precipitated Ca2+-phosphate, thus permitting cells to survive situations of Ca2+ emergency. The uptake process was found to have very low affinity for Ca2+: since the bulk concentration of Ca2+ in the cytoplasm is in the low to mid-nM range, it became increasingly difficult to postulate a role of mitochondria in the regulation of cytoplsmic Ca2+. A number of findings had nevertheless shown that energy linked Ca2+ transport occurred efficiently in mitochondria of various tissues in situ. The paradox was only solved in the 1990s, when it was found that the concentration of Ca2+ in the cytoplasm is not uniform: perimitochondrial micropools are created by the agonist-promoted discharge of Ca2+ from vicinal stores in which the concentration of Ca2+ is high enough to activate the low affinity mitochondrial uniporter. Mitochondria thus regained center stage as important regulators of cytoplasmic Ca2+ (not only of their own internal Ca2+). Their Ca2+ uptake systems was found to react very rapidly to cytoplasmic Ca2+ demands, even in the 150-200 msec time scale of processes like the contraction and relaxation of heart. An important recent development in the area of mitochondrial Ca2+ transport is its involvement in the disease process. Ca2+ signaling defects are now gaining increasing importance in the pathogenesis of diseases, e.g., neurodegenerative diseases. Since mitochondria have now regained a central role in the regulation of cytoplasmic Ca2+, dysfunctions of their Ca2+ controlling systems have expectedly been found to be involved in the pathogenesis of numerous disease processes. 相似文献
19.
Skeletal muscle phosphorylase kinase (PhK) is a Ca2+-dependent enzyme complex, (αβγδ)4, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca2+-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation-contraction-energy production triad.” Although the Ca2+-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca2+-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca2+ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca2+ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca2+-binding proteins troponin C and CaM. 相似文献
20.
Isolated mussel mitochondria produced a less pronounced transient stimulation of respiration upon the addition of Ca2+ in a reaction medium containing Pi and a slower rate of Ca2+ transport compared to rat liver mitochondria. The initial rates of Ca2+ transport in the absence of Pi were more similar and both types of mitochondria possessed a sigmoidal relationship between the initial rate of Ca2+ transport and the free Ca2+ concentration . Ruthenium red produced an equal maximal inhibition of the initial rate of Ca2+ transport in both types of mitochondria but mussel mitochondria were rather more resistant to the inhibitor. The major difference found was that approximately 15 nmoles La3+ mg protein?1 was required to produce maximal inhibition of the initial rate of Ca2+ transport in mussel mitochondria compared to approximately 1.0 nmole La3+ mg protein?1 in rat liver mitochondria. It is concluded that mussel mitochondria possess a comparable Ca2+ transporter to vertebrate mitochondria and possible reasons for resistance to La3+ are discussed. 相似文献