心肌肥厚大鼠心肌细胞核三磷酸肌醇受体的特征

为了研究细胞核三磷酸肌醇受体在心肌肥厚中的作用,制备了腹主动脉缩窄大鼠心肌肥厚模型、用差速离心和密度梯度离心提纯心肌细胞核,以［3H］IP3为配基,采用放射受体分析心肌细胞核膜IP3R与其配体的最大结合容量(Bmax)和解离常数(Kd)。大鼠心肌细胞核上存在IP3R、CaM和PKC激动剂PMA,能显著抑制该受体与IP3的结合(P<0.05);核外［Ca2+］也能剂量依赖的抑制细胞核IP3R与IP3的结合。腹主动脉缩窄术后4周,大鼠心肌显著肥大,伴有明显的血流动力学异常,其心肌细胞核IP3R的Bmax和Kd与对照组比较分别增加1.217和2.149倍(P<0.01)。心肌细胞核上存在IP3R,并受CaM和PMA及核外［Ca     

甲胎蛋白受体的性质及其所介导的cAMP-PKA信号转导途径

研究HeLa细胞膜上甲胎蛋白 (alpha fetoprotein ,AFP)受体的存在情况及其介导的信号转导 .先用Na[12 5I]标记AFP ;标记的AFP和培养的HeLa细胞结合 ,Scatchard法和受体 配体结合法分析受体数目 ;再用放射免疫结合法分析在百日咳毒素 (pertussistoxin ,PTX)预处理前后AFP对细胞内环腺苷酸 (cAMP)浓度及细胞内蛋白激酶A(proteinkinaseA ,PKA)活性变化的影响 .在HeLa细胞膜表面存在 2种不同解离平衡常数 (Kd)的AFP受体 ,Kd1=5 2pmol L(2 10 0位点 细胞 ) ;Kd2 =2 3nmol L (114 0 0位点 细胞 ) .在AFP(2 0mg L)作用下 ,HeLa细胞内cAMP浓度变化及PKA活性的改变为与对照组比较 ,用PTX预处理前cAMP浓度升高 2 6 7% ,PKA活性增高 10 3 2 % ;用PTX预处理后升高 86 % ,PKA活性增高 2 5 3% .抗甲胎蛋白单克隆抗体可阻断AFP对细胞cAMP浓度和PKA活性的影响 .结果证明 ,在HeLa细胞膜上有 2种不同解离平衡常数的甲胎蛋白受体存在 ,受体有可能通过cAMP PKA途径介导信号转导 .     

压力超负荷性心肌肥厚大鼠心肌细胞核钙转运的改变

通过腹主动脉缩窄(abdominalaorticcoarctation ,AAC)心肌肥厚大鼠模型制备、差速离心提纯心肌细胞核、酶学方法测定Ca2 +-ATPase活性、45Ca2 +同位素法测定核钙摄取和荧光分光光度计测定细胞核内自由钙浓度 ,初步揭示压力超负荷心肌肥厚大鼠心肌细胞核钙转导异常的环节。结果发现 :心肌细胞核上存在具有[Ca2 +]和ATP依赖性的高亲和力Ca2 +-ATPase ,以[Ca2 +]依赖的方式摄取45Ca2 +,并呈先升高后降低趋势。AAC术后4周大鼠心肌显著肥厚 ,伴有明显的血流动力学异常 ,与对照组比较 ,AAC大鼠心肌细胞核Ca2 +-ATPase活性减少51.93 %(p<0.001) ,但核45Ca2 +摄入量(核外[Ca2 +]浓度为800 -1600nmol/L时)和核内[Ca2 +](核外[Ca2 +]浓度为0 -1000nmol/L时)均明显增加(p<0.05) ;正常组离体心肌细胞核Ca2 +摄取受PKA刺激(p<0.05) ,而被PKC抑制剂和CaM抑制剂显著抑制(p<0.05) ,AAC大鼠心肌细胞核Ca2 +摄取仅受CaM抑制剂抑制(p<0.01) ,而PKA和PKC抑制剂对其无明显影响(p>0.05)。结论为心肌肥厚时 ,心肌细胞核Ca2 +转运系统及其磷酸化调节可能发生改变。     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca2+浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 ]i 升高 ,即 PKC和 Ca2 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca~(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

猪脑突触质膜糖皮质激素膜结合位点的测定

利用放射配体结合法,测定了猪脑六个不同脑区突触质膜糖皮质激素膜结合位点(GCM-BS)的最大结合量(Bmax)和平衡解离常数(Kd)。结果表明:猪的大脑、小脑、中脑、丘脑、下丘脑和尾状核的突触质膜GCMBS的Kd都在200nmol/L左右,提示它们对[~3H]皮质酮的亲和力基本相同;而各脑区内GCMBS的Bmax相差较大,从218.98fmol/mg蛋白至486.95fmol/mg蛋白不等,说明不同脑区内所含有的GCMBS的数量不同。     

甲硫氨酸脑啡肽和抗δ阿片受体抗体对小鼠骨髓瘤细胞信号传递系统的作用

为了探讨阿片肽与细胞表面受体结合后所产生的生物效应及其机制 ,用不同浓度甲硫氨酸脑啡肽 ( MENK)及抗 δ阿片受体单克隆抗体处理小鼠的骨髓瘤细胞 ( NS- 1 ) ,然后测定蛋白激酶A( PKA) ,蛋白激酶 C( PKC)活性及三磷酸肌醇 ( IP3 )含量 .研究结果表明 ,NENK可升高细胞胞浆及胞膜 PKA的活性 ,且这一作用可被抗δ阿片受体抗体所拮抗 .MENK对 PKC影响呈双向反应 ,0 .1 μmol/L MENK可以升高胞浆 PKC活性 ,但却明显降低胞膜 PKC活性 ;在 MENK浓度为 1 0μmol/L时则情况刚好相反 .1 μmol/L的 MENK可明显降低胞浆及胞膜 PKC活性 ,抗体可拮抗这种下调作用 .MENK可降低细胞内 IP3 的含量 ,且这一作用可被抗δ阿片受体抗体所拮抗 .由此可以推论 :MENK在与 δ阿片受体结合后 ,可以经过多种信号传导系统来调节细胞功能 ,从而产生不同的生物效应 .     

非NMDA受体的细胞内调控及其机制

非NMDA受体是AMPA受体和KA受体的统称,其亚基上的胞内C末端上有很多磷酸化脱磷酸化及胞内蛋白的结合位点,这些作用位点与非NMDA受体的调控密切相关。如PKA,PKC,PKG,CaMKⅡ和PTK等蛋白激酶均可调节受体的活性,另外,胞内的一些骨架蛋白和信号蛋白也可与非NMDA受体结合而影响其功能,本文着重综述非NMDA受全的细胞内调控及其机制。     

心肌肌钙蛋白Ⅰ的磷酸化与非磷酸化调节

由于心肌肌钙蛋白复合体Ⅰ亚基(Troponin Ⅰ,TnⅠ)特殊的分子结构,使其在心肌收缩过程中起"分子开关"的重要作用.心肌TnⅠ具有6个磷酸化位点,第23/24位丝氨酸残基可被蛋白激酶A(PKA)、蛋白激酶D(PKD)和蛋白激酶G(PKG)磷酸化,发挥正性肌力作用;第43/45位丝氨酸残基以及第144位酪氨酸残基可被蛋白激酶C(PKC)磷酸化,可能主要起负性肌力作用;蛋白激活激酶(PAK)磷酸化第149位丝氨酸残基后的作用尚待探明.另外,经蛋白水解酶calpain降解含磷酸化位点的片段,产生去磷酸化作用;亦可通过降解一些特定片段来改变TnⅠ空间构象,引起非磷酸化调节作用.     

内皮素-1通过L-型钙通道的Ca~(2+)内流和钙致钙释放诱导心肌细胞内[Ca~(2+)]的增加

本研究利用fura-2-AM荧光成像和膜片钳技术,发现内皮素-1(Endothelin-1,ET-1)可显著提高大鼠分离心肌细胞内钙离子水平([Ca2+]i),激活心肌细胞钙通道.ETA受体阻滞剂BQ123能够消除ET-1提高[Ca2+]i的效应,而ETB受体阻滞剂BQ788对该效应无影响.用ryanodine受体阻断剂ryanodine(10 μmol/L)预处理,可以使ET-1诱导的[Ca2+]i的增加抑制46.7%.蛋白激酶A(PKA)的抑制剂、蛋白激酶C(PKC)的抑制剂和血管紧张素Ⅱ一型受体(AT1 receptor)的抑制剂都能够抑制ET-1诱导的[Ca2+]i的增加.本研究发现ET-1能够提高全细胞L-型钙通道电流的幅度,增加L-型钙通道单通道的开放概率.并且BQ123完全阻止了ET-1诱导的L-型钙通道开放概率增加的效应.本研究证明了ET-1通过一系列机制调节钙超载,包括L-型钙通道的激活,钙致钙释放(CICR),ETA受体,PKC,PKA和血管紧张素Ⅱ一型受体也参与到了这个途径中.     

败血症大鼠心肌核被膜和肌浆网ryanodine受体的变化

为观察败血症时心肌肌浆网（ＳＲ）和核被膜（ＮＥ）的ｒｙａｎｏｄｉｎｅ受体的变化,采用结扎及穿刺盲肠（ＣＬＰ）制作败血症动物模型,用密度梯度离心分离ＳＲ和ＮＥ,用放射配体结合法研究ｒｙａｎｏｄｉｎｅ受体的特征。结果表明,大鼠早期败血症（ＣＬＰ后９ｈ）时,ＳＲ的ｒｙａｎｏｄｉｎｅ受体的最大结合（Ｂｍａｘ）增加２３％,ＮＥ的ｒｙａｎｏｄｉｎｅ受体的Ｂｍａｘ则增加１倍,二者比值降低３９％（Ｐ＜００１）;在晚期败血症（ＣＬＰ后１８ｈ）时,ＳＲｒｙａｎｏｄｉｎｅ受体的Ｂｍａｘ降低了３８％,ＮＥ的ｒｙａｎｏｄｉｎｅ受体的Ｂｍａｘ增加１６倍,二者比值降低７６％;ＳＲ和ＮＥｒｙａｎｏｄｉｎｅ受体的离解常数无显著改变。败血症时,ＳＲｒｙａｎｏｄｉｎｅ受体早期上调,晚期下调,而ＮＥｒｙａｎｏｄｉｎｅ受体均上调,这些变化可能与休克时相有关。     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.     

Functional properties of ryanodine receptors from rat dorsal root ganglia

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (Bmax) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (Kd) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+-dependent and displayed the classical long-lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+-dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.     

Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures

Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Direct binding of verapamil to the ryanodine receptor channel of sarcoplasmic reticulum.

Radioligand binding experiments and single channel recordings demonstrate that verapamil interacts with the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum of rabbit skeletal muscle. In isolated triads, verapamil decreased binding of [3H]Ryanodine with an IC50 of approximately 8 microM at an optimal pH 8.5 and pCa 4.3. Nitrendipine and d-cis-diltiazem did not interfere with binding of [3H]Ryanodine to triads, suggesting that the action of verapamil does not involve the dihydropyridine receptor. Single channel recordings showed that verapamil blocked Ca2+ release channels by decreasing open probability, duration of open events, and number of events per unit time. A direct interaction of verapamil with the ryanodine receptor peptide was demonstrated after purification of the approximately 400 kDa receptor protein from Chaps-solubilized triads. The purified receptor displayed high affinity for [3H]Ryanodine with a Kd of approximately 5 nM and a Bmax of approximately 400 pmol/mg. Verapamil and D600 decreased [3H]Ryanodine binding noncompetitively by reducing the Bmax. Thus the presence of binding sites for phenylalkylamines in the Ca2+ release channel was confirmed. Verapamil blockade of Ca2+ release channels may explain some of the paralyzing effects of phenylalkylamines observed during excitation-contraction coupling of skeletal muscle.     

Two types of inositol trisphosphate binding in cardiac microsomes.

Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.     

The cardiac sarcoplasmic reticulum calcium-release channel: modulation of ryanodine binding and single-channel activity

[3H]Ryanodine binding to a preparation of isolated cardiac sarcoplasmic reticulum has been investigated. A method is reported which produces a very high level of specific binding. Scatchard analysis of binding up to 50 nM ryanodine yields data which infer a single class of binding sites with a Kd of 1.4 nM and a Bmax of 9.7 pmol/mg protein. Micromolar calcium is the principal activating ligand and its effects on binding are modulated by ligands which similarly affect the activity of single calcium-release channels incorporated into artificial planar phospholipid bilayers. The benzimidazole drug, sulmazole, is able to stimulate ryanodine binding in the presence of sub-activating calcium concentrations. Ryanodine binds to the native channel only when it is in its open state and stimulation of maximal ryanodine binding is achieved by ligands which are insufficient to produce full single-channel activation. A model is proposed which relates the modulation of ryanodine binding to the behaviour of single channels.     

Functional characterisation of the ryanodine receptor purified from sheep cardiac muscle sarcoplasmic reticulum

Sheep cardiac muscle sarcoplasmic reticulum ryanodine receptors have been isolated by density-gradient centrifugation following solubilisation with the zwitterionic detergent, CHAPS. The functional state of the receptor complex has been assessed by quantification of [3H]ryanodine binding and by characterisation of single-channel conductance and gating properties following reconstitution into unilamellar proteo-liposomes and incorporation into planar phospholipid bilayers. A method of solubilisation is described which yields a receptor displaying high-affinity [3H]ryanodine binding (Kd 2.8 nM, Bmax 352 pmol/mg protein) and which functions as a cation-selective, ligand-regulated channel under voltage clamp conditions. Previous reports of channel activity of purified rabbit skeletal and canine cardiac muscle ryanodine receptors describe a range of sub- or variable-conductance events. In contrast, the sheep cardiac ryanodine receptor-channels isolated using the optimal conditions described in this report consistently display a single open state conductance with either Ca2+ or K+ as the charge carrying species.     

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.