Department of Neurobiology and Behavior, University of California, Irvine, CA.
Biophys J. 2013 Dec 3;105(11):2474-84. doi: 10.1016/j.bpj.2013.10.028.
Puffs are localized, transient elevations in cytosolic Ca(2+) that serve both as the building blocks of global cellular Ca(2+) signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP3Rs), whose openings are coordinated by Ca(2+)-induced Ca(2+) release (CICR). We utilized total internal reflection fluorescence imaging of Ca(2+) signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ∼66% of opening following opening of an initial "trigger" channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca(2+)-inhibited IP3Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP3-which would not be subject to earlier Ca(2+)-inhibition-also showed wide variability, indicating that mechanisms such as stochastic variation in IP3 binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size.
脉冲是细胞溶质 Ca(2+) 的局部、瞬时升高，既是全局细胞 Ca(2+)信号的组成部分，也是自身的局部信号。它们源自聚集的肌醇 1,4,5-三磷酸受体/通道 (IP3Rs)，其开放受 Ca(2+)-诱导的 Ca(2+)释放 (CICR) 协调。我们利用具有单通道分辨率的神经母细胞瘤细胞内 Ca(2+)信号的全内反射荧光成像来阐明决定脉冲触发、幅度、动力学和空间扩展的机制。我们发现，簇中的任何给定通道在初始“触发”通道打开后，其打开的平均概率约为 66%，并且脉冲触发的概率随着簇中通道数量的增加而急剧增加 (簇大小)。平均脉冲幅度与簇大小成正比，但单个幅度变化很大，即使在簇大小相似的部位，涉及簇中任意数量通道的事件比例也相似。Ca(2+)-抑制型 IP3R 的数量的随机变化可能导致在一个部位重复脉冲的幅度变化，但连续脉冲的幅度是不相关的，尽管我们观察到了脉冲间隔和脉冲幅度之间的统计相关性。光释放在 IP3 后引发的初始脉冲也表现出很大的变异性，这表明 IP3 结合和 CICR 招募通道的随机变化等机制进一步决定了脉冲幅度。随着脉冲幅度的增加，脉冲的平均终止时间延长，这与给定的平均开放时间后通道的独立关闭一致，但我们发现终止时间与簇大小之间没有相关性，而与脉冲幅度无关。脉冲的空间范围随着幅度的增加而增加，并且幅度相似的脉冲具有相似的宽度，而与簇大小无关。
