Ca²⁺ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca²⁺. Intracellular Ca²⁺ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca²⁺ handling properties by combining the ‘added buffer’ approach [1] with perforated patch-clamp recordings [2]. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca²⁺ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca²⁺ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca²⁺ handling properties, including immobile as well as mobile Ca²⁺ buffers.

Ca ²⁺作为多种细胞过程的重要细胞内信号发挥作用。这些过程由游离胞质 Ca ²⁺的受控时空动力学选择性激活。细胞内Ca ²⁺动力学受到许多细胞参数的调节。在这里，我们通过将“添加缓冲”方法 [1] 与穿孔膜片钳记录 [2] 相结合，建立了一种确定神经元 Ca ²⁺处理特性的新方法。由于添加缓冲液方法通常采用标准全细胞配置来进行浓度控制的 Ca ²⁺指示剂加载，因此它只能可靠地估计细胞内 Ca ²⁺缓冲液的固定部分。此外，在长时间的全细胞记录过程中，细胞内信号通路的关键成分被冲走，导致细胞恶化。通过将添加缓冲液方法与穿孔膜片钳记录相结合，可以避免这些问题，从而可以精确量化细胞 Ca ²⁺处理特性，包括固定和移动 Ca ²⁺缓冲液。