Electrophysiological Analysis of SAN Cells

SAN cells were harvested in custom-made chambers with glass bottoms for cell attachment and superfused with Tyrode solution warmed at 36 °C before recording. All electrophysiological signals, APs and ion currents, were measured by perforated patch-clamp technique and recorded using a Multiclamp 700 A patch clamp amplifier connected to Digidata 1550B interface (Molecular Devices). Electrodes had a resistance of 3–4 MΩ when filled with a solution containing (in mM): 80 K-aspartate, 50 KCl, 1 MgCl₂, 2 CaCl₂, 5 EGTA, 5 HEPES, and 3 ATP-Na (adjusted to pH 7.2 with KOH). Perforated patch-clamp was performed by adding 30 µM β-escin to the intracellular solution. Seal resistances were in the range of 2–5 GΩ. Perfusion of pre-warmed (36 °C) experimental solutions was achieved by using a multi-MPRE8 heating pen (Cell Micro Controls). AP parameters were measured as previously described (see Supplementary Table S1 inset in ref. [27 (link)]). For ion current measurements, dormancy or firing of SAN cells at baseline were recorded in current-clamp for at least 30 s before switching to voltage-clamp.