KINETICS OF CALCIUM-CHANNEL OPENING BY INOSITOL 1,4,5-TRISPHOSPHATE
KINETICS OF CALCIUM-CHANNEL OPENING BY INOSITOL 1,4,5-TRISPHOSPHATE
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DOI：
10.1021/bi00453a004
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发表时间：
1990-01-09
期刊：
影响因子：
2.9
通讯作者：
STRYER, L
中科院分区：
文献类型：
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作者：
MEYER, T;WENSEL, T;STRYER, L
用快速混合技术测量了IP 3对细胞内Ca 2+的亚秒级动员，以确定细胞在受体触发的钙尖峰期间如何实现胞质[Ca 2 +]的快速上升。在透化的大鼠嗜碱性白血病细胞中，在11 ℃下，C，超过80%的0.7 fmol的Ca 2 +/细胞螯合的ATP驱动泵可以释放的IP 3。在加入饱和（1 μ M）IP 3后，一半的储存Ca 2+在200 ms内释放。通量速率在120 nM IP 3下为半最大值。从满载商店的钙释放是高度合作的，希尔系数在2-40纳米范围内大于3。通道开放的延迟时间与[IP 3]成反比，从100 nM IP 3时的150 ms增加到15 nM时的1 s，表明通道开放的限速步骤是IP 3结合。需要多个结合步骤来解释所观察到的通道开放的延迟和非指数特性。提出了一个简单的模型，其中四个IP 3分子的绑定到相同的和独立的网站导致通道开放。该模型与KD = 18 nM，kon = 1.2 × 10 - 4的数据吻合良好。108 M-1 s-1，koff = 2.2 s-1。. apprx.结合的IP 3的1-s交换时间表明通道门控位点不同于具有约100-s交换时间，这是以前用放射性标记的IP 3发现的。. apprx. [Ca 2 +]对IP 3水平快速增加的1-s响应时间可以解释观察到的钙峰上升时间。
The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11.degree. C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-drive pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 .mu.M) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 .times. 108 M-1 s-1, and koff = 2.2 s-1. The .apprx. 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having .apprx. 100-s exchange times that were previously found with radiolabeled IP3. The .apprx. 1-s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.