Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.     

Calmodulin and troponin C: affinity chromatographic study of divalent cation requirements for troponin I inhibitory peptide (residues 104-115), mastoparan and fluphenazine binding

The different conformations induced by the binding of Mg2+ or Ca2+ to troponin C (TnC) and calmodulin (CaM) results in the exposure of various interfaces with potential to bind target compounds. The interaction of TnC or CaM with three affinity columns with ligands of either the synthetic peptide of troponin I (TnI) inhibitory region (residues 104-115), mastoparan (a wasp venom peptide), or fluphenazine (a phenothiazine drug) were investigated in the presence of Mg2+ or Ca2+. TnC and CaM in the presence of either Ca2+ or Mg2+ bound to the TnI peptide 104-115. The cation specificity for this interaction firmly establishes that the TnI inhibitory region binds to the high affinity sites of TnC (most likely the N-terminal helix of site III) and presumably the homologous region of CaM. Mastoparan interacted strongly with both proteins in the presence of Ca2+ but, in the presence of Mg2+, did not bind to TnC and only bound weakly to CaM. Fluphenazine bound to TnC and CaM only in the presence of Ca2+. When the ligands interacted with either proteins there was an increase in cation affinity, such that TnC and CaM were eluted from the TnI peptide or mastoparan affinity column with 0.1 M EDTA compared with the 0.01 M EDTA required to elute the proteins from the fluphenazine column. The interaction of these ligands with their receptor sites on TnC and CaM require a specific and spatially correct alignment of hydrophobic and negatively charged residues on these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

从拟南芥表达文库中筛选钙调素结合蛋白cDNA克隆

以生物素标记的钙调素为探针,筛选拟南芥λZAPⅡ表达文库,分离得到9个阳性克隆。融合蛋白的Westem印迹分析表明,这些阳性克隆确是编码钙调素结合蛋白的(图2,3),并且其中Y9等8个克隆编码的融合蛋白与钙调素有依赖于钙离子的结合(图3B);而唯独Y7编码的融合蛋白与钙调素的结合,与CaMBP-10一样,不依赖于钙离子的存在(图3A)。     

Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C

Binding of trifluoperazine (TFP), a phenothiazine tranquilizer, to porcine brain calmodulin (CaM) and rabbit skeletal muscle troponin C (Tn C) was measured by an automated high-performance liquid chromatography binding assay using a molecular sieving column; 10 micrograms of either protein per injection is sufficient for determining TFP binding, and results are comparable to those obtained by equilibrium dialysis. Very little binding was observed to either protein in the absence of Ca2+ while in the presence of Ca2+ both proteins bind 4 equiv of TFP. Other characteristics of TFP binding however are different for each protein. For CaM, half-maximal binding occurs at 5.8 microM TFP, the Hill coefficient is 0.82, and the fit of the data to the Scatchard equation is consistent with four independent TFP-binding sites. Binding of one melittin displaces two TFP from CaM. Thus, there are two recognizable classes of TFP-binding sites: those that are displaced by melittin and those that are not. TFP causes an increase in the Ca2+ affinity of CaM, and three Ca2+ must be bound to CaM for TFP binding to occur. The studies also yielded a measure of the intrinsic affinity of three of CaM's Ca2(+)-binding sites that is in agreement with previous reports. For troponin C, half-maximal binding occurs at 16 microM TFP, the Hill coefficient is 1.7, and the data best fit the Adair equation for four binding sites. The measured constants K1, K2, K3, and K4 were 2.5 X 10(4), 6.6 X 10(3), 5.8 X 10(5), and 2.0 X 10(5) M-1, respectively, in 1 mM Ca2+ and were similar when Mg2+ was additionally included. TFP also increases troponin C's Ca2+ affinity, and it is the low-affinity, Ca2(+)-specific binding sites that are affected. These studies yielded a measure of the intrinsic affinity of these Ca2(+)-binding sites that is in agreement with previous measurements.     

Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca(2+), B. emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca(2+) channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca(2+)-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii.     

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.     

Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle.

The effects of calmodulin (CaM) on inositol 1,4,5-trisphosphate (InsP3) 3-kinase activity in pig aortic smooth muscle were examined. The cytosol fraction of muscle cells, containing 1.2-2.0 micrograms of CaM/mg of cytosol protein (thus 0.12-0.2%, w/w), showed a Ca2+-dependent InsP3 3-kinase activity, and there was no further activation by exogenous addition of CaM purified from dog brain. (NH4)2SO4 fractionation of the cytosol fraction revealed that a 20-60%-satd.-(NH4)2SO4 fraction was rich in the enzyme activity, and the activity without exogenous CaM was still dependent on Ca2+, although the CaM content in this fraction was minute (0.013-0.016%, w/w). The kinase activity observed in the absence of exogenous CaM became insensitive to Ca2+ when a 20-60%-satd.-(NH4)2SO4 fraction was applied to a DEAE-cellulose column, but exogenous addition of CaM increased the enzyme activity from 80-120 to 450 pmol/min per mg of protein, with addition of 10 microM free Ca2+. A fraction separated by DEAE-cellulose chromatography was applied to a CaM affinity column. The kinase activity was retained on the column in the presence of Ca2+, and was eluted by lowering the free Ca2+ concentration by adding EGTA. These results directly show that CaM activates InsP3 3-kinase activity and the enzyme becomes sensitive to Ca2+.     

Glyoxalase I from Brassica juncea is a calmodulin stimulated protein.

Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1. 5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg2+) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca2+/CaM). In the presence of Ca2+ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca2+ dependent mobility shift on denaturing gels. Its Ca2+ binding was confirmed by Chelex-100 assay and gel overlays using 45CaCl2. Glyoxalase I was activated by over 7-fold in the presence of Ca2+ (25 microM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 microM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in the presence of Mg2+ and Ca2+; however, magnesium alone also showed glyoxalase I activation by CaM.     

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

The synthesis and reaction of a specific affinity label for the hydrophobic drug-binding domains of calmodulin

An affinity-labeling reagent for the two hydrophobic drug-binding domains of calmodulin has been prepared and its reaction with calmodulin characterized. The reagent, 10-(3-propionyloxysuccinimide)-2-(trifluoromethyl)phenothiazine, was shown to be very specific labeling reagent for these domains. Its specificity was demonstrated by the following observations. 1) Previous reports have shown that Ca2+ is required for phenothiazine binding to calmodulin, and here we show that the affinity-labeling reagent reacts with and inactivates calmodulin in the presence of Ca2+, but not in its absence. 2) Inclusion of trifluoperazine, fluphenazine, W-7, or 10-(3-aminopropyl)-2-(trifluoromethyl)phenothiazine in the reaction mixture protected calmodulin from inactivation by the reagent. 3) Inactivation by the reagent yielded calmodulin that was no longer retained on a phenothiazine-Sepharose column under conditions in which unreacted calmodulin was retained. 4) The measured stoichiometry of the reaction in the presence of excess reagent was 2.1 mol of reagent per mol of calmodulin which agrees well with previous reports of two high-affinity phenothiazine-binding sites on calmodulin. 5) The stoichiometry of the reaction was further confirmed by tryptic peptide maps which show two phenothiazine-labeled peptides unique to the fully reacted protein. 6) The spectral properties of the reagent, while attached to calmodulin, change in the presence of Ca2+ in a manner consistent with the known effects of Ca2+ binding by calmodulin on these hydrophobic domains. The specificity of the reagent makes it useful for further characterization of these hydrophobic binding domains on calmodulin.     

Trifluoperazine binding to calmodulin: a shift reagent 43CA NMR study

43Ca NMR experiments of Ca2+ binding to calmodulin (CaM) were performed in the presence and absence of the calmodulin antagonist trifluoperazine (TFP). By making use of the shift reagent Dy(PPP)(7-) (a 1:2 complex of DyCl3 and Na5P3O10) we have succeeded in separating the 43Ca resonances of protein-bound Ca2+ and free Ca2+ in the otherwise unresolved spectra. This experimental strategy has allowed us to demonstrate unequivocally that the affinity of CaM for Ca2+ is markedly increased in the presence of TFP. Thus Ca2+ is not liberated from the protein upon addition of TFP as had been suggested based on earlier 43Ca NMR experiments (Shimuzu, T., Hatano, M., Nagao, S. and Nozawa, Y. (1982), Biochem. Biophys. Res. Comm. 106, 1112-1118).     

Functional properties of covalent beta-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation

The 31-residue neuropeptide porcine beta-endorphin was shown to inhibit the Ca2+-dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to beta-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular beta-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. We found that relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking beta-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked beta-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. In contrast, the 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 beta-endorphin 13-31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+-dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium dependence of the interaction between calmodulin and anthrax edema factor

Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity. CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated. Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis. Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM. The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter. In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity. As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation. This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state.     

Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene

Both normal chicken calmodulin (CaM) and a CaM-like mutant protein have been expressed in bacteria, isolated and evaluated with respect to several physical and biological properties. The mutant CaM is derived from a CaM-like gene that lacks intervening sequences and probably evolved from a CaM-processed gene (Stein, J. P., Munjaal, R. P., Lagacé, L., Lai, E. C., O'Malley, B. W., and Means, A. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6485-6489). The mutant CaM protein contains 16 of the 19 amino acids encoded by the CaM-like gene. Normal chicken CaM produced in bacteria is identical to rat CaM by all criteria tested except that it is not trimethylated. The protein product of the CaM-like gene has been termed CaML and exhibits properties which are very similar to CaM despite the presence of 16 amino acid substitutions. CaML binds Ca2+ as evidenced by Ca2+-dependent binding to phenothiazine- and phenyl-Sepharose affinity resins and a Ca2+-dependent electrophoretic mobility shift which is similar to but distinct from CaM. CaML cross-reacts with a monospecific CaM antibody and has an immunodilution curve which is identical to bacterially synthesized CaM. Finally, CaML can maximally activate rat brain phosphodiesterase but with altered kinetic parameters as compared to CaM. These data suggest that the nucleotide substitutions in the putative CaM processed gene are not random but are selected to retain CaM-like functions in the encoded protein. Such a mechanism may exist for other processed genes.     

Regulation of cAMP concentration by calmodulin-dependent cyclic nucleotide phosphodiesterase

Bovine brain contains two major calmodulin (CaM) dependent phosphodiesterase isozymes which are homodimeric proteins with subunit molecular masses of 60 and 63 kilodaltons (kDa), respectively. The 60-kDa subunit isozyme can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme affinity towards CaM. The phosphorylation is blocked by Ca2+ and CaM and reversed by the CaM-stimulated phosphatase (calcineurin). The 63-kDa subunit isozymes can also be phosphorylated, but in this case by a CaM-dependent protein kinase(s). This phosphorylation is also accompanied by a decrease in the isozyme affinity towards CaM and can be reversed by the CaM-dependent phosphatase. Analysis of the complex regulatory properties of the phosphodiesterase isozymes has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaM-dependent phosphodiesterase isozymes play key roles in this signal coupling phenomenon.     

果蝇TRP离子通道C端一个新钙调蛋白结合位点的鉴定和分析

瞬时受体电位(TRP)通道是一类钙离子透过性的阳离子通道蛋白家族,参与了视觉、味觉、温度感受等重要的生物学过程。之前的研究表明,钙离子既能够正反馈也能够负反馈地调节瞬时受体电位通道的活性,而这种调节可能是通过钙调蛋白（calmodulin,CaM）与TRP通道的相互作用来进行的。为了阐明这一调控机制,我们首先需要对钙调蛋白与瞬时受体电位通道之间的相互作用进行详细的生化研究。在此项研究中,通过大肠杆菌表达系统,表达和纯化了果蝇瞬时受体电位通道羧基末端不同长短的蛋白片段,并发现了一个新的钙调蛋白结合位点。通过快速蛋白液相色谱、静态光散射以及等温量热滴定技术,鉴定了这一钙调蛋白结合位点与果蝇瞬时受体电位通道之间的相互作用,发现它们在钙离子依赖的条件下,可以形成亲和力非常强的稳定的蛋白复合物（解离常数在01～1微摩尔范围）。此外,通过合成多肽的方法,鉴定了果蝇瞬时受体电位通道913～939片段为该钙调蛋白结合位点的核心区域。最后,通过突变实验,进一步明确了果蝇瞬时受体电位通道922位的酪氨酸以及923位的缬氨酸为其钙调蛋白结合位点的关键氨基酸。总而言之,本研究发现和鉴定了果蝇瞬时受体电位通道上一个新的钙依赖的钙调蛋白结合位点,这一发现将为研究瞬时受体电位通道的体内功能提供生化基础,为阐明钙离子通过钙调蛋白调节瞬时受体电位通道的分子机制做出贡献。