A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle

Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.     

High-capacity Ca2+ binding of human skeletal calsequestrin

Calsequestrin, the major calcium storage protein in both cardiac and skeletal muscle, binds large amounts of Ca(2+) in the sarcoplasmic reticulum and releases them during muscle contraction. For the first time, the crystal structures of Ca(2+) complexes for both human (hCASQ1) and rabbit (rCASQ1) skeletal calsequestrin were determined, clearly defining their Ca(2+) sequestration capabilities through resolution of high- and low-affinity Ca(2+)-binding sites. rCASQ1 crystallized in low CaCl(2) buffer reveals three high-affinity Ca(2+) sites with trigonal bipyramidal, octahedral, and pentagonal bipyramidal coordination geometries, along with three low-affinity Ca(2+) sites. hCASQ1 crystallized in high CaCl(2) shows 15 Ca(2+) ions, including the six Ca(2+) ions in rCASQ1. Most of the low-affinity sites, some of which are μ-carboxylate-bridged, are established by the rotation of dimer interfaces, indicating cooperative Ca(2+) binding that is consistent with our atomic absorption spectroscopic data. On the basis of these findings, we propose a mechanism for the observed in vitro and in vivo dynamic high-capacity and low-affinity Ca(2+)-binding activity of calsequestrin.     

Luminal Ca2+ regulation of single cardiac ryanodine receptors: insights provided by calsequestrin and its mutants

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 muM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below approximately 0.5 mM.     

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.     

Assembly of the sarcoplasmic reticulum. Synthesis of calsequestrin and the Ca2+ + Mg2+ -adenosine triphosphatase on membrane-bound polyribosomes.

Membrane-bound and free polyribosomes were isolated from skeletal muscle of neonatal rats and messages were translated in a rabbit reticulocyte lysate treated with Ca2+ -dependent nuclease to reduce endogenous messenger translation. Newly synthesized calsequestrin and adenosine triphosphatase (ATPase) sere isolated by antibody precipitation, followed by separation of the precipitates in SDS-polyacrylamide gels. Radioactivity in calsequestrin and the ATPase were counted in gel slices. Calsewuestrin and the ATPase were both found to be synthesized on membrane-bound polyribosomes. Since calsequestrin is a glycoprotein, localized in Golgi regions in early stages of muscle cell differentiation, it is probable that its synthesis follows the pathway for synthesis of secreted proteins except that its destination is the luminal space of a cellular organelle. The disposition of the ATPase during synthesis is, as yet, unknown.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.     

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.     

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.     

Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors

The plasma membrane Na+/Ca2+ exchanger (NCX) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes. Binding of Na+ and Ca2+ ions to its large cytosolic loop regulates ion transport of the exchanger. We determined the solution structures of two Ca2+ binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD), form the regulatory exchanger loop. CBD1 and CBD2 are very similar in the Ca2+ bound state and describe the Calx-beta motif. Strikingly, in the absence of Ca2+, the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a 7-fold higher affinity for Ca2+, this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.     

Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin

Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.     

Evolution of mechanisms of Ca2+-signalization. Role of Ca2+ in regulation of specialized cell functions

The review describes peculiarities of Ca²⁺-signalization in electro-excitable cells of higher eukaryotes. The light has been shed on problems of Ca²⁺-dependent mechanisms of regulation of muscle contractility and of neuronal synaptic plasticity in the higher vertebrate animals. A particular attention has been paid to analysis of contribution of such poorly studied components of Ca²⁺-signalization as non-selective TRPC-channels, Orai channels, sensory STIM1 proteins, Ca²⁺-controlled K⁺-channels of large and small conductance, and neuronal Ca²⁺-sensors (NCS).     

Ca2+-dependent regulation of cyclic-AMP phosphodiesterase by parvalbumin.

Carp parvalbumin has been shown to activate rat brain phosphodiesterase in a Ca2+-dependent manner. The concentration of Ca2+ required for half-maximal stimulation is 1.4 X 10(-7) M, whereas rat testis Ca2+-dependent regulator (CDR) of phosphodiesterase required 1.2 X 10(-6) M Ca2+. The difference in the slopes of the two curves demonstrated that the activation induced by parvalbumin was not the result of a small contamination by CDR. In addition, it has been shown that Ca2+ binding to parvalbumin parallels its activation of phosphodiesterase. These data suggest that Ca2+ must bind to a single specific metal binding site before phosphodiesterase can be fully activated.     

Evolution of mechanisms of Ca2+-signaling. Role of Ca2+ in regulation of fundamental cell functions

The review considers mechanisms of Ca2+-dependent regulation of cell growth, differentiation, and apoptosis in cells of the higher eukaryotes by modulation of the signal Ras-MAPK pathway.