Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C

We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T. The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites. Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+. In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites. Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results. Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299-304). Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.     

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.     

The dynamics of luminal depletion and the stochastic gating of Ca2+-activated Ca2+ channels and release sites

Single channel models of intracellular calcium (Ca(2+)) channels such as the 1,4,5-trisphosphate receptor and ryanodine receptor often assume that Ca(2+)-dependent transitions are mediated by constant background cytosolic [Ca(2+)]. This assumption neglects the fact that Ca(2+) released by open channels may influence subsequent gating through the processes of Ca(2+)-activation or inactivation. Similarly, the influence of the dynamics of luminal depletion on the stochastic gating of intracellular Ca(2+) channels is often neglected, in spite of the fact that the sarco/endoplasmic reticulum [Ca(2+)] near the luminal face of intracellular Ca(2+) channels influences the driving force for Ca(2+), the rate of Ca(2+) release, and the magnitude and time course of the consequent increase in cytosolic domain [Ca(2+)]. Here we analyze how the steady-state open probability of several minimal Ca(2+)-regulated Ca(2+) channel models depends on the conductance of the channel and the time constants for the relaxation of elevated cytosolic [Ca(2+)] and depleted luminal [Ca(2+)] to the bulk [Ca(2+)] of both compartments. Our approach includes Monte Carlo simulation as well as numerical solution of a system of advection-reaction equations for the multivariate probability density of elevated cytosolic [Ca(2+)] and depleted luminal [Ca(2+)] conditioned on each state of the stochastically gating channel. Both methods are subsequently used to study the role of luminal depletion in the dynamics of Ca(2+) puff/spark termination in release sites composed of Ca(2+) channels that are activated, but not inactivated, by cytosolic Ca(2+). The probability density approach shows that such minimal Ca(2+) release site models may exhibit puff/spark-like dynamics in either of two distinct parameter regimes. In one case, puffs/spark termination is due to the process of stochastic attrition and facilitated by rapid Ca(2+) domain collapse [cf. DeRemigio, H., Smith, G., 2005. The dynamics of stochastic attrition viewed as an absorption time on a terminating Markov chain. Cell Calcium 38, 73-86]. In the second case, puff/spark termination is promoted by the local depletion of luminal Ca(2+).     

Relationships between Ca2+ release, Ca2+ cycling, and Ca2+-mediated permeability changes in mitochondria

Ruthenium red and/or EGTA prevent cyclic uptake and release of Ca2+ in mitochondria. These compounds inhibit but do not prevent the swelling of liver mitochondria induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. Ruthenium red and/or EGTA have complex effects on the release rate of Ca2+ and other cations induced by t-butyl hydroperoxide or N-ethylmaleimide. To determine the relationship between permeability changes and Ca2+ release in the absence of Ca2+ cycling, a novel method of data collection and analysis is developed which allows the relative time courses of Ca2+ release and Mg2+ release or swelling to be accurately and quantitatively compared. This method eliminates errors in time course comparisons which arise from the aging of mitochondrial preparations and allows data from different preparations to be directly contrasted. Using the method, it is shown that permeability changes caused by Ca2+-releasing agents are not secondary effects arising from Ca2+ cycling between uptake and release carriers. In the absence of Ca2+-cycling inhibitors, Ca2+ release induced by t-butyl hydroperoxide or N-ethylmaleimide is, in part, carrier-mediated. In the presence of EGTA and ruthenium red, Ca2+ release induced by either agent is mediated solely by the permeability pathway. No differences are apparent in the solute selectivity of the inner membrane permeability defect induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. A novel type of Ca2+ release from energized liver mitochondria is reported. This release is induced by EGTA, occurs in the absence of other releasing agents or nonspecific permeability changes, and is rapid (greater than or equal to 50 nmol/min/mg protein).     

Ca2+ regulation of interactions between endoplasmic reticulum chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.     

Characterization by Hg2+ of two different pathways for mitochondrial Ca2+ release

The addition of Hg2+ to loaded kidney mitochondria induces the fast release of the accumulated cation. The Ca2+-efflux reaction exhibits kinetics characteristics that depend on the extent of the binding of Hg2+ to the membrane. At high levels of Hg2+ bound (approx. 11 nmol/mg), Ca2+ efflux rate is highly insensitive to the temperature of incubation, and the efflux seems to be directly related to the internal free Ca2+ concentration. At these levels of bound Hg2+, accumulated Sr2+ is released with characteristics similar to those observed with Ca2+. At lower levels of Hg2+ binding (2.5 nmol/mg), the efflux reaction is highly dependent on the incubation temperature and on the internal free Ca2+ concentration; under these conditions Sr2+ is not released. NAD(P)H oxidation as induced by the low Hg2+ concentration is inhibited at the lower temperatures. Radiolabeled Hg2+ incorporates into two clearly defined regions of membrane proteins separated through sodium dodecyl sulfate gel electrophoresis. One of the regions corresponds to proteins of apparent high molecular mass (i.e., 150 kDa), and the other to proteins with apparent molecular masses of 37-25 kDa. Mitochondria incubated with 2 microM 203Hg2+ incorporate the radionuclide in proteins that have molecular masses of around 41 and 26 kDa. The results indicate that, depending on the amount of Hg2+ bound to the inner membrane, two clearly distinct Ca2+ release mechanisms can be distinguished.     

Osmoregulation of atrial myocytic ANP release: osmotransduction via cross-talk between L-type Ca2+ channel and SR Ca2+ release

Hyperosmolality has been known to increase ANP release. However, its physiological role in the regulation of atrial myocytic ANP release and the mechanism by which hyperosmolality increases ANP release are to be defined. The purpose of the present study was to define these questions. Experiments were performed in perfused beating rabbit atria. Hyperosmolality increased atrial ANP release, cAMP efflux, and atrial dynamics in a concentration-dependent manner. The osmolality threshold for the increase in ANP release was as low as 10 mosmol/kgH2O (approximately 3%) above the basal levels (1.55 +/- 1.71, 17.19 +/- 3.11, 23.15 +/- 5.49, 54.04 +/- 11.98, and 62.00 +/- 13.48% for 10, 20, 30, 60, and 100 mM mannitol, respectively; all P < 0.01). Blockade of sarcolemmal L-type Ca2+ channel activity, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (-13.58 +/- 4.68% vs. 62.00 +/- 13.48%, P < 0.001) and cAMP efflux but not atrial dynamics. Blockade of the Ca2+ release from the sarcoplasmic reticulum, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (13.44 +/- 7.47% vs. 62.00 +/- 13.48%, P < 0.01) and dynamics but not cAMP efflux. Blockades of Na+-K+-2Cl- cotransporter, Na+/H+ exchanger, and Na+/Ca2+ exchanger had no effect on hyperosmolality-induced increase in ANP release. The present study suggests that hyperosmolality regulates atrial myocytic ANP release and that the mechanism by which hyperosmolality activates ANP release is closely related to the cross-talk between the sarcolemmal L-type Ca2+ channel activity and sarcoplasmic reticulum Ca2+ release, possibly inactivation of the L-type Ca2+ channels.     

Intra- and extraluminal sarcoplasmic reticulum membrane regulatory sites for Ca2(+)-induced Ca2+ release.

A heavy skeletal muscle sarcoplasmic reticulum (SR) fraction was actively loaded stepwise with calcium until Ca2(+)-induced Ca2+ release occurred. The total Ca2+ load, T1, at which release occurred is postulated to be regulated by an intraluminal, low-affinity receptor. After obtaining T1, the critical concentration of Ca2+ required extraluminally (T2) was determined. T1 averaged 58.6 +/- S.D., 6.9 nmol Ca2+/mg SR and T2 averaged 2.14 +/- S.D., 0.24 microM. Both T1 and T2 were increased by Mg2+ and decreased by caffeine. Ruthenium red increased T2 more than T1 while ryanodine had no effect on T1 but markedly increased T2. The results suggest that two Ca2+ regulatory sites may be functional for Ca2(+)-induced Ca2+ release from SR.     

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).     

Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

The binding constants, K₁ and K₂, and the number of Ca²⁺ ions in each of the two high affinity sites of Ca²⁺-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K₁ and K₂, it was found that two hydrogen ions are released for each Ca²⁺ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K_s, for the binding of Ca²⁺ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K_s and its pH dependence with those of K₁ and K₂ showed that the site corresponding to K_s is to be identified with that of K₂. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca²⁺ that causes the blue to purple color change.

Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

     

Stationary Ca2+ nanodomains in the presence of buffers with two binding sites

We examine closed-form approximations for the equilibrium Ca²⁺ and buffer concentrations near a point Ca²⁺ source representing a Ca²⁺ channel, in the presence of a mobile buffer with two Ca²⁺ binding sites activated sequentially and possessing distinct binding affinities and kinetics. This allows us to model the impact on Ca²⁺ nanodomains of realistic endogenous Ca²⁺ buffers characterized by cooperative Ca²⁺ binding, such as calretinin. The approximations we present involve a combination or rational and exponential functions, whose parameters are constrained using the series interpolation method that we recently introduced for the case of simpler Ca²⁺ buffers with a single Ca²⁺ binding site. We conduct extensive parameter sensitivity analysis and show that the obtained closed-form approximations achieve reasonable qualitative accuracy for a wide range of buffer’s Ca²⁺ binding properties and other relevant model parameters. In particular, the accuracy of the derived approximants exceeds that of the rapid buffering approximation in large portions of the relevant parameter space.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites

The active site residues in calpain are mis-aligned in the apo, Ca(2+)-free form. Alignment for catalysis requires binding of Ca2+ to two non-EF-hand sites, one in each of the core domains I and II. Using domain swap constructs between the protease cores of the mu and m isoforms (which have different Ca2+ requirements) and structural and biochemical characterization of site-directed mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding to this domain as shown by the crystal structure of D106A muI-II. However, at elevated Ca2+ concentrations this mutant still forms the double salt bridge that links the two Ca2+ sites and becomes nearly as active as muI-II. Elimination of the bridge in E333A muI-II has a more drastic effect on enzyme action, especially at low Ca2+ concentrations. Domain II Ca2+ binding appears essential, because Ca(2+)-coordinating side-chain mutants E302R and D333A have severely impaired muI-II activation and activity. The introduction of mutations into the whole heterodimeric enzyme that eliminate the salt bridge or Ca2+ binding to domain II produce similar phenotypes, suggesting that the protease core Ca2+ switch is crucial and cannot be overridden by Ca2+ binding to other domains.     

Use of the coulombic interactions of the lanthanide series to identify two classes of Ca2+ binding sites in mitochondria

The degree of inhibition of respiration-dependent vs respiration-independent Ca²⁺ binding by rat liver mitochondria by different members of the lanthanide family was used to establish the existence of two different classes of Ca²⁺ binding sites. The distinction is based on the differences in cation:site interactions between the two classes of sites and the members of the lanthanide series. Lanthanide inhibition of respiration-dependent Ca²⁺ uptake suggests that the binding site is specific for the calcium ion. Those members of the lanthanide family whose ionic radii are nearer that of Ca²⁺ are the best inhibitors. The inhibition of respiration-independent Ca²⁺ binding is much different, indicating non-specific cation absorption.     

Direct coupling between arachidonic acid-induced Ca2+ release and Ca2+ entry in HEK293 cells

Arachidonic acid (AA) modulates intracellular Ca2+ signaling via Ca2+ release or/and Ca2+ entry. However, the mechanism underlies either process is unknown; nor is it clear as to whether the two processes are mechanistically linked. By using Fura2/AM, we found that AA induced mobilization of internal Ca2+ store and an increment in Ca2+, Mn2+ and Ba2+ influx in HEK293 cells. The AA-mediated Ca2+ signaling was not due to AA metabolites, and insensitive to capacitative Ca2+ entry inhibitors. Interestingly, isotetrandrine and Gd3+ inhibited both AA-induced Ca2+ release and Ca2+ entry in a concentration-dependent manner without affecting Ca2+ discharge caused by carbachol, caffeine, or thapsigargin. Additionally, similar pattern of inhibition was observed with tetracaine treatment. More importantly, the three compounds exhibited almost equal potent inhibition of AA-initiated Ca2+ release as well as Ca2+ influx. Therefore, this study, for the first time, provides evidence for a direct coupling between AA-mediated Ca2+ release and Ca2+ entry.