Analysis of neuronal Ca²⁺ handling properties by combining perforated patch clamp recordings and the added buffer...

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.