Patch-clamp recordings of SAN cells

Patch-clamp recordings of SAN cells was carried out as previously described⁵². Cells were harvested in custom-made chambers with glass bottoms for cell attachment. Cells were then superfused with Tyrode’s solution warmed at 36 °C before recording. Voltage-gated Ca²⁺ currents (I_Ca) were recorded using standard whole cell patch-clamp configuration as previously described^23,24. Extracellular recording solution contained (mM): 135 tetraethylammonium chloride (TEA-Cl), 10 4-aminopyridine (4-AP), 1 MgCl₂, 0.03 tetrodotoxin (TTX), 1 g/L Glucose, 2 CaCl₂, 10 Hepes, (adjusted to pH = 7.2 with TEAOH). Electrodes had a resistance of 3 MΩ and were filled with a solution containing (in mM): 125 Cs⁺-aspartate, 20 TEA-Cl, 1.2 CaCl₂, 5 Mg-ATP, 0.1 Li₂-GTP, 5 EGTA, and 10 HEPES (pH adjusted to 7.2 with CsOH)^23,24. Seal resistances were in the range of 2–5 GΩ. Pacemaker activity of SAN cells was recorded under perforated patch conditions by adding 50 μM β-Escin to the pipette intracellular solution^23,35. Patch-clamp electrodes had a resistance of 3–5 MΩ when filled with an intracellular solution containing (in mM): 130 K⁺-aspartate, 10.0 NaCl; 2 ATP-Na⁺ salt, 6.6 creatine phosphate, 0.1 GTP-Mg²⁺, 0.04 CaCl₂ (pCa = 7.0), and 10.0 HEPES–KOH (adjusted to pH 7.2 with KOH). Perfusion of pre-warmed (36 °C) experimental solutions was performed by using a multi-MPRE8 heating pen (Cell Micro Controls,). Data acquisition was performed using a Multiclamp 700A patch-clamp amplifier connected to Digidata 1550B interface (Molecular Devices).