Watras J, Moraru I, Costa D J, Kindman L A
University of Connecticut Health Center, Farmington 06032.
Biochemistry. 1994 Nov 29;33(47):14359-67. doi: 10.1021/bi00251a050.
Quantal calcium release is a novel paradigm for second messenger signal transduction which provides spatial and temporal control of calcium release from intracellular stores by inositol 1,4,5-trisphosphate (InsP3). We have proposed a mechanism to account for this phenomenon [Kindman, L. A., & Meyer, T. (1993) Biochemistry 32, 1270-1277], which hypothesized the existence of five channels, each with a different affinity for InsP3. As a direct test of this hypothesis, InsP3 binding to microsomes from RBL cells was examined under conditions similar to those used for calcium release. Scatchard analyses performed under a variety of conditions indicates the presence of high affinity (KD = 0.9 +/- 0.3 nM) and low affinity (KD = 47 +/- 5 nM) InsP3 binding sites. The low affinity sites are more prevalent, constituting 82 +/- 5% of the total. Both sites are identified in the presence and absence of MgATP. Moreover, both sites are selective for InsP3 over InsP4, through high concentrations of InsP4 displace InsP3 from each site (with inhibition constants of 16 and 267 nM InsP4, respectively). The relative abundance of the two InsP3 binding sites is Ca2+ dependent. An increase in Ca2+ from 0.1 to 0.5 microM results in the apparent conversion of a portion of the low affinity sites into high affinity sites into high affinity sites. Ca2+ (0.5 microM) also increased the KD of the low affinity InsP3 binding site. Given the presence of both high and low affinity InsP3 binding sites, two simple mathematical models describing both the kinetics of calcium release and quantal calcium release from RBL cells were developed.(ABSTRACT TRUNCATED AT 250 WORDS)
量子钙释放是第二信使信号转导的一种新范式，它通过肌醇1,4,5 -三磷酸（InsP3）对细胞内钙库的钙释放提供空间和时间控制。我们提出了一种解释这种现象的机制[金德曼，L.A.，& 迈耶，T.（1993年）《生物化学》32卷，1270 - 1277页]，该机制假设存在五个通道，每个通道对InsP3具有不同的亲和力。作为对这一假设的直接检验，在类似于用于钙释放的条件下，检测了InsP3与RBL细胞微粒体的结合。在各种条件下进行的斯卡查德分析表明存在高亲和力（KD = 0.9 ± 0.3 nM）和低亲和力（KD = 47 ± 5 nM）的InsP3结合位点。低亲和力位点更为普遍，占总数的82 ± 5%。在有和没有MgATP的情况下都能识别出这两种位点。此外，通过高浓度的InsP4从每个位点取代InsP3（抑制常数分别为16和267 nM InsP4），这两种位点对InsP3的选择性都高于InsP4。两种InsP3结合位点的相对丰度依赖于Ca2+。Ca2+从0.1 μM增加到0.5 μM会导致一部分低亲和力位点明显转化为高亲和力位点。Ca2+（0.5 μM）也增加了低亲和力InsP3结合位点的KD。鉴于存在高亲和力和低亲和力的InsP3结合位点，开发了两个简单的数学模型来描述RBL细胞中钙释放和量子钙释放的动力学。（摘要截短至250字）
