Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca²⁺. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca²⁺ is linked.     

6周有氧运动对高脂膳食的ApoE敲除小鼠骨骼肌钙调控的影响*

目的: 探讨6周有氧运动对高脂膳食的载脂蛋白E(ApoE)基因敲除小鼠骨骼肌肌浆网钙调控蛋白的影响。方法: 25只9周龄ApoE敲除小鼠(ApoE KO)随机选取5只ApoE KO小鼠进行最大跑速测试(以初始速度为4.8 m/min,坡度为0°,持续5 min后,每3 min速度增加1.2 m/min,直至力竭,最后速度为最大跑速,最大跑速的测试结果为(27.0±2.4)m/min,剩余20只ApoE KO小鼠随机分为ApoE KO小鼠高脂膳食组(KO)和ApoE KO小鼠高脂膳食+有氧运动组(KE),每组10只,同时以10只9周龄野生型C57BL/6J小鼠作为空白对照组(WT)。高脂饲料成分:脂肪含量为21%(w/w),胆固醇含量为1.5%(w/w)。KE组适应性训练1周后开始运动干预,运动方案为:40%最大跑速(10.8 m/min),运动时间40 min/d,频率每周3 d,共计6周。待末次运动后48 h,所有小鼠麻醉后经心脏穿刺处死后迅速分离双侧腓肠肌;可见光比色法检测骨骼肌Ca²⁺浓度;Western blot法检测小鼠骨骼肌肌浆网钙调控蛋白RyR、CaM、CaMKⅡ、SERCA1、SERCA2蛋白表达。结果: 与WT组相比,KO组小鼠骨骼肌Ca²⁺浓度显著降低(P＜0.01),骨骼肌肌浆网钙释放蛋白RyR、CaMKⅡ和钙回收蛋白SERCA1、SERCA2均显著降低(P＜ 0.05),但CaM蛋白无显著变化;与KO组相比,KE组小鼠骨骼肌Ca²⁺浓度和骨骼肌肌浆网钙回收蛋白SERCA1、SERCA2均显著升高(P＜0.05),但骨骼肌肌浆网钙释放蛋白RyR、CaM、CaMKⅡ蛋白表达均无显著性差异。结论: 高脂膳食可使ApoE敲除小鼠骨骼肌Ca²⁺浓度降低、肌浆网钙释放作用和钙回收作用减弱,6周有氧运动训练能够显著提高其Ca²⁺浓度、促进肌浆网钙回收作用。     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes

We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or -myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca²⁺-ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations. cardiac myocytes; cell-specific expression; adenovirus vectors; calcium transport     

Differential regulation of SR calcium transporters by thyroid hormone in rat atria and ventricles

Thyroid hormone exerts positive inotropic effects on the heart mediated in part by its regulation of calcium transporter proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2), phospholamban (PLB), and Na(+)/Ca(2+) exchanger (NCX). To further understand the potential cardiac chamber-specific effects of thyroid hormone action, we compared the triiodo-L-thyronine (T(3)) responsiveness of calcium transporter proteins in atrial versus ventricular tissues. Rats were rendered hypothyroid by ingestion of propylthiouracil, and a subgroup of animals was treated with T(3) for 7 days (7 microg/day by constant infusion). Atrial and left ventricular (LV) tissue homogenates were analyzed for expression of SERCA2, PLB, and NCX proteins by Western blot analysis. SERCA2 protein significantly decreased by 50% in hypothyroid LV and was normalized by T(3) treatment. In contrast, SERCA2 protein in atria was unaltered in the hypothyroid state. PLB protein expression significantly increased by 1.6- and 5-fold in the hypothyroid LV and atria, respectively, and returned to euthyroid levels with T(3) treatment. Expression of NCX protein showed a greater response to T(3) treatment in atria tissue than in ventricular tissue. Sarcoplasmic reticulum calcium cycling is determined in part by the ratio of SERCA2 to PLB. This ratio was sixfold higher in the atria compared with LV, suggesting that PLB may play a minor role in the regulation of SERCA2 function in normal atria. We conclude that calcium transporter proteins are responsive to thyroid hormone in a chamber-specific manner, with atria showing a greater change in protein content in response to T(3). The differential effect on atria may account for the occurrence of atrial rather than ventricular arrhythmias in response to even mild degrees of thyrotoxicosis.     

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.     

Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction.     

Amino acid sequence of alpha chain of sarcoplasmic calcium binding protein obtained from shrimp tail muscle

The isotypes of sarcoplasmic Ca2+ binding protein (SCP) were purified from shrimp tail muscle. SCP exists in a dimeric form. One sample of shrimp contained only alpha A chain, whereas another contained alpha B and beta chains, and a heterodimer of alpha B beta which was not analyzed precisely. The amino acid sequences of the two alpha chains were determined. The two alpha chains are composed of 190 and 192 amino acid residues, respectively. The sequences of the two alpha chains differed in only four amino acids out of 192 residues. The sequences indicate that the alpha chain has three Ca2+-binding sites which are common to EF-hand type Ca2+-binding protein. In the absence of added Ca2+ and Mg2+, the amounts of bound Ca2+ in alpha A, alpha B, and beta chains were 3.0, 3.3, and 2.4 mol/22,000 g protein, respectively. Thus, it is suggested that all three isotypes of shrimp SCP have three Ca2+-binding sites which have high affinity to Ca2+. The sequence homology of shrimp SCP with other EF-hand type Ca2+-binding proteins is very low. The protein having the greatest homology with this SCP was cod parvalbumin; the sequence homology is 18%.     

Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer

To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 Å. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.     

Conditional increase in SERCA2a protein is able to reverse contractile dysfunction and abnormal calcium flux in established diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by reduced cardiac contractility independent of vascular disease. A contributor to contractile dysfunction in the diabetic heart is impaired sarcoplasmic reticulum function with reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) pump activity, leading to disturbed intracellular calcium handling. It is currently unclear whether increasing SERCA2a activity in hearts with existing diabetic cardiomyopathy could still improve calcium flux and contractile performance. To test this hypothesis, we generated a cardiac-specific tetracycline-inducible double transgenic mouse, which allows for doxycycline (DOX)-based inducible SERCA2a expression in which DOX exposure turns on SERCA2a expression. Isolated cardiomyocytes and Langendorff perfused hearts from streptozotocin-induced diabetic mice were studied. Our results show that total SERCA2a protein levels were decreased in the diabetic mice by 60% compared with control. SERCA2a increased above control values in the diabetic mice after DOX. Dysfunctional contractility in the diabetic cardiomyocyte was restored to normal by induction of SERCA2a expression. Calcium transients from diabetic cardiomyocytes showed a delayed rate of diastolic calcium decay of 66%, which was reverted toward normal after SERCA2a expression induced by DOX. Global cardiac function assessed in the diabetic perfused heart showed diminished left ventricular pressure, rate of contraction, and relaxation. These parameters were returned to control values by SERCA2a expression. In conclusion, we have used mice allowing for inducible expression of SERCA2a and could demonstrate that increased expression of SERCA2a leads to improved cardiac function in mice with an already established diabetic cardiomyopathy in absence of detrimental effects.     

Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.     

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure

The altered calcium binding activity of calmodulins (CaM) with point mutations can be restored toward that of wild type CaMs by the formation of a complex between CaM and a CaM binding sequence. Three different site-specific mutations resulted in selective effects on the apparent stoichiometry and affinity of CaM for calcium, with maintenance of the ability to activate myosin light chain kinase. The effects on calcium binding, however, were suppressed when the mutant CaMs were complexed with RS20, a peptide analog of a myosin light chain kinase CaM binding site. The mutations included: 1) a Glu----Ala mutation at two phylogenetically conserved calcium ligands in the second (E67A-CaM) and fourth (E140A-CaM) sites; and 2) a Ser----Phe mutation at residue 101 (S101F-CaM) which affects ion channel regulation. The mutant CaMs bind 4 calciums in the absence of magnesium, but two sites have approximately 60- to 300-fold weaker binding than wild-type CaM (SYNCAM CaM). E67A-CaM and E140A-CaM bound only two calciums and S101F-CaM bound 4 calciums in the presence of magnesium. E67A-CaM and E140A-CaM recovered the ability to bind 4 calcium ions in the presence of the RS20 CaM binding peptide. These results are consistent with models in which the calcium binding activity of CaM within a supramolecular complex is different from purified CaM and raise the possibility that the selective functional effects of in vivo mutations in the calcium binding sites of CaM might be partially due to the ability of some CaM binding proteins to select and utilize CaM conformations with calcium ligation structures different from the so-called canonical EF-hand.     

Burn trauma alters calcium transporter protein expression in the heart.

We have shown previously that burn trauma produces significant cardiac dysfunction, which is first evident 8 h postburn and is maximal 24 h postburn. Because calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism by which burn injury may cause cardiac abnormalities is via calcium dyshomeostasis. We hypothesized that major burn injury alters cardiomyocyte calcium handling through changes in calcium transporter expression. Sprague-Dawley rats were given either burn injury or no burn injury (controls). Cardiomyocyte intracellular calcium and sodium were quantified at various times postburn by fura 2-AM or sodium-binding benzofuran isophthalate fluorescent indicators, respectively. In addition, hearts freeze-clamped at various times postburn (2, 4, 8, and 24 h) were used for Western blot analysis using antibodies against the sarcoplasmic reticulum calcium-ATPase (SERCA), the L-type calcium-channel, the ryanodine receptor, the sodium/calcium exchanger, or the sodium-potassium-ATPase. Intracellular calcium levels were elevated significantly 8-24 h postburn, and intracellular sodium was increased significantly 4 through 24 h postburn. Expression of SERCA was significantly reduced 1-8 h postburn, whereas L-type calcium-channel expression was diminished 1 and 2 h postburn (P < 0.05) but returned toward control levels 4 h postburn. Ryanodine receptor protein was significantly reduced at 1 and 2 h postburn, returning to baseline by 4 h postburn. Sodium/calcium exchanger expression was significantly elevated 2 h postburn but was significantly reduced 24 h postburn. An increase in sodium-potassium-ATPase expression occurred 2-24 h postburn. These data confirm that burn trauma alters calcium transporter expression, likely contributing to cardiomyocyte calcium loading and cardiac contractile dysfunction.     

Inhibition of CaMKII Does Not Attenuate Cardiac Hypertrophy in Mice with Dysfunctional Ryanodine Receptor

In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca²⁺/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2^ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2^ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2^ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2^ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca²⁺ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2^ADA/ADA mice.     

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting

Calmodulin (CaM) is a highly conserved calcium (Ca²⁺) binding protein that transduces Ca²⁺ signals into downstream effects influencing a range of cellular processes, including Ca²⁺ homeostasis. The present study explores CaM expression when Ca²⁺ homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca²⁺ ATPase (PMCA) known to regulate intracellular Ca²⁺ through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca²⁺ flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.     

Designing calcium-sensitizing mutations in the regulatory domain of cardiac troponin C

Cardiac troponin C belongs to the EF-hand superfamily of calcium-binding proteins and plays an essential role in the regulation of muscle contraction and relaxation. To follow calcium binding and exchange with the regulatory N-terminal domain (N-domain) of human cardiac troponin C, we substituted Phe at position 27 with Trp, making a fluorescent cardiac troponin C(F27W). Trp(27) accurately reported the kinetics of calcium association and dissociation of the N-domain of cardiac troponin C(F27W). To sensitize the N-domain of cardiac troponin C(F27W) to calcium, we individually substituted the hydrophobic residues Phe(20), Val(44), Met(45), Leu(48), and Met(81) with polar Gln. These mutations were designed to increase the calcium affinity of the N-domain of cardiac troponin C by facilitating the movement of helices B and C (BC unit) away from helices N, A, and D (NAD unit). As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C(F27W) approximately 2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6-8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C(F27W) and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mm). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.     

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.