Inositol 1,4,5-trisphosphate (IP₃) evokes Ca²⁺ release through IP₃ receptors (IP₃Rs) to generate both local Ca²⁺ puffs arising from concerted openings of clustered IP₃Rs and cell-wide Ca²⁺ waves. Imaging Ca²⁺ puffs with single-channel resolution yields information on the localization and properties of native IP₃Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP₃Rs. Here, we used TIRF and light-sheet microscopy to image Ca²⁺ puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP₃R type 1, 2, or 3. Photorelease of the IP₃ analog i-IP₃ in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca²⁺ flux was similar among isoforms, indicating that clusters of different IP₃R isoforms contain comparable numbers of active channels. Our results show that all three IP₃R isoforms cluster to generate local Ca²⁺ puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells.

肌醇1,4,5-三磷酸酯（IP ₃）引起通过IP ₃受体（IP ₃ Rs）释放Ca ²⁺，从而产生由簇状IP ₃ Rs和整个细胞内Ca ^2+的一致开放引起的局部Ca ²⁺抽吸。波浪。用单通道分辨率对Ca ²⁺抽吸进行成像可得到完整IP细胞中天然IP ₃ Rs的定位和特性的信息，但是由于细胞表达IP ₃ Rs的三种结构和功能不同同工型的比例不同，因此解释非常复杂。在这里，我们使用TIRF和光片显微镜对Ca ²⁺进行成像通过CRISPR-Cas9技术生成的HEK-293细胞系中的粉扑可仅表达IP ₃ R类型1、2或3。在所有三个细胞系中IP ₃类似物i-IP _3的光释放引起了粉扑，其平均振幅非常相似，时间特征和空间范围。此外，同工型之间的单通道Ca ²⁺通量相似，表明不同IP ₃ R同工型的簇包含相当数量的活性通道。我们的结果表明，所有三个IP ₃ R同工型均会簇生局部Ca ²⁺ 与来自体外电生理研究的不同性质的发现相反，在完整细胞中显示出相似的电导和门控动力学。