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

Simon Hess , Christophe Pouzat (1) , Lars Paeger , Andreas Pippow , Peter Kloppenburg

1 MAP5 - UMR 8145 - Mathématiques Appliquées Paris 5

Ca 2+ 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 2+ . Intracellular Ca 2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca 2+ 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 Ca 2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca 2+ 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 2+ handling properties, including immobile as well as mobile Ca 2+ buffers.

Mots clés: Added buffer approach β-Escin Calcium-binding ratio Calcium buffering Cerebellum Fura-2 Midbrain Perforated patch clamp

Domaines: Applications [stat.AP]; Neurobiologie

Simon Hess, Christophe Pouzat, Lars Paeger, Andreas Pippow, Peter Kloppenburg. Analysis of neuronal Ca 2+ handling properties by combining perforated patch clamp recordings and the added buffer approach. Cell Calcium, 2021, 97, pp.102411. ⟨10.1016/j.ceca.2021.102411⟩. ⟨hal-03031583⟩