Functional characterization of the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed in HEK293 cells
Functional characterization of the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed in HEK293 cells
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DOI：
10.1074/jbc.272.39.24234
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发表时间：
1997-09-26
影响因子：
4.8
通讯作者：
Zhang, L
中科院分区：
文献类型：
--
作者：
Chen, SRW;Li, XL;Zhang, L
为了研究哺乳动物3型兰尼碱受体（RyR 3）的通道特性，我们用逆转录-聚合酶链反应从兔子宫中克隆了RyR 3 cDNA，并在大肠杆菌中表达。HEK 293细胞中的cDNA，免疫印迹研究表明克隆的RyR 3在分子大小和免疫反应性上与天然哺乳动物RyR 3没有区别。荧光Ca ~（2+）指示剂Fluo 3检测结果表明，克隆的RyR 3在HEK 293细胞中具有咖啡因和鱼龙碱敏感的Ca ~（2+）释放通道功能。在250 mM KCl中，K+电导为777皮西门子，Ca ~（2+）电导为137皮西门子，pCa ~（2+）/pK ~（+）比值为6.3，开放时间常数约为1.16 ms。Ca ~（2+）在100 nM左右激活通道，在10 mM左右失活。单独Ca ~（2+）能完全激活克隆的RyR 3，钙调素在低Ca ~（2+）浓度下激活克隆的RyR 3，但在低Ca ~（2+）浓度下抑制通道。克隆的RyR 3被ATP、咖啡因和高氯酸盐激活，被Mg ~（2+）和钌红抑制，并被ryanodine修饰，环ADP-核糖似乎不影响克隆的RyR 3的单通道活性。从兔骨骼肌Ryanodine受体克隆RyR 3的最显着的差异是在门控动力学，最大激活程度的Ca 2+，和敏感性Ca 2+失活。本研究的结果提供了初步了解哺乳动物RyR 3的单通道特性。
To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed! the cDNA inn HEK293 cells, Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca2+ release channel in HEK293 cells, Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+ conductance of 777 picosiemens in 250 mM KCl and a Ca2+ conductance of 137 picosiemens inn 250 mM CaCl2 and displayed a pCa(2+)/pK(+) ratio of 6.3 and an open time constant of about 1.16 ms, The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nM and inactivated at about 10 mM. Ca2+ alone was able to activate the cloned RyR3 fully, Calmodulin activated the cloned RyR3 at low Ca2(+) concentrations but inhibited the channel at low Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine, cyclic ADP-ribose did not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maximal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3.