High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels.
High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels.
复制标题
从心脏连接肌浆网囊泡纯化的高分子量蛋白质是兰尼碱敏感的钙通道。
DOI：
10.1161/01.res.64.4.779
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发表时间：
1989
影响因子：
20.1
通讯作者：
Jones,LR
中科院分区：
文献类型：
--
作者：
Rardon,DP;Cefali,DC;Mitchell,RD;Seiler,SM;Jones,LR
心脏高分子量蛋白/兰尼碱受体从交界肌质网膜纯化至同质，并显示出大电导钙通道活性。将连接肌浆网中的高分子量蛋白质溶解在两性离子洗涤剂中，并通过尺寸排阻色谱法和随后的蔗糖密度梯度离心进行纯化。纯化的蛋白质表现出表观Mr = 400,000-350,000，并以4.6 nM的Kd和140-280 pmol/mg蛋白质的Bmax结合[3H]ryanodine。高分子量蛋白质在掺入平面脂质双层后表现出二价阳离子通道活性。确定了两种渠道类型。大电导通道的斜率电导为 96 +/- 13 pS，Erev 为 42 +/- 9 mV (n = 5)；小电导通道的斜率电导为 5.5 +/- 1 pS [1.0 microM cis CaCl2； 50 mM 反式 Ba(OH)2]。将顺式钙从 1 microM 减少到 1 nM，将大电导通道开放时间从 7 +/- 1% 减少到 0.1%（保持电位，-100 mV）。向顺室添加 ATP (1 mM) 将通道开放时间从 6 +/- 1% 增加到 52 +/- 4%（保持电位，-100 mV）； 10 nM 兰尼碱增加了 96 pS 通道的开放时间百分比，100 µM 兰尼碱减少了 96 pS 通道的开放时间百分比，但不改变单一通道电导。大电导通道与在天然犬心脏连接肌浆网囊泡中检测到的钙释放通道相似。我们的数据表明，兰尼碱受体、钙释放通道和高分子量蛋白质都是相同的蛋白质，含有钙、ATP 和兰尼碱的变构调节位点。
The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.