Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

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     

Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

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.     

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

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.     

Monitoring the Structural Behavior of Troponin and Myoplasmic Free Ca Concentration during Twitch of Frog Skeletal Muscle

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.     

The exchange of Ca2+-receptive protein complex (troponin) in the myofibrils of fast and slow skeletal muscles

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.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

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.     

Ca release from mitochondria induced by prooxidants

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⁺.     

Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria

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.     

Calcium binding to cardiac troponin C

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⁺.     

Structure–function relationships of molluscan troponin T revealed by limited proteolysis

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.     

Calcium uptake mechanisms of mitochondria

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.     

The effect of ruthenium red on the uptake and release of Ca 2+ by mitochondria

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.     

The Ca2+-Transport System of Yeast (Endomyces magnusii) Mitochondria: Independent Pathways for Ca2+ Uptake and Release

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.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

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.     

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

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.     

Effects of prostaglandins on the interaction of Ca2+ with mitochondria

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⁺.     

The interaction of La 3+ with mitochondria in relation to respiration-coupled Ca 2+ transport

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.     

The fateful encounter of mitochondria with calcium: How did it happen?

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.     

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle

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.     

The mechanism of calcium transport in mitochondria isolated from the marine mussel, Mytilus edulis (L.)

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 $\text{(‘Km’ ? 5μM)}$. 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.