Analysis of neuronal Ca 2+ handling properties by combining perforated patch clamp recordings and the added buffer approach

Ca 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 Ca . Intracellular Ca dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca 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 Ca indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca 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 Ca handling properties, including immobile as well as mobile Ca buffers.