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bioRxiv · 10.1101/2020.07.10.164202

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

Simon Hess·Christpohe Pouzat·Lars Paeger·Andreas Pippow·Peter Kloppenburg
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Abstract

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 (Neher and Augustine, 1992) with perforated patch-clamp recordings (Horn and Marty, 1988). 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.Competing Interest StatementThe authors have declared no competing interest.

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Simon Hess, Christpohe Pouzat, Lars Paeger, Andreas Pippow, Peter Kloppenburg. 2020-07-10. Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach. https://doi.org/10.1101/2020.07.10.164202

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