Whole-cell voltage clamp electrophysiological recordings

VDCCs, K_ATP and K_V currents were recorded from dispersed, primary mouse β-cells with the whole-cell voltage clamp technique³¹. Patch electrodes were pulled with tip resistance between 3–4 μΩ. For VDCC recordings, patch electrodes were loaded with intracellular solution containing (in mM): 102 CsCl, 10 TEA chloride, 0.1 tolbutamide, 10 EGTA, 1 MgCl₂, 3 Na₂ATP, 5 HEPES, pH 7.4, adjusted with CsOH. Whole β-cell seals (>1 giga-ohm) were made in KRB buffer in the presence of 3 mM glucose followed by perifusion with a modified, high Ca²⁺-containing KRB buffer of the following composition (in mM): 82 NaCl, 20 TEA chloride, 0.1 tolbutamide, 30 CaCl₂, 5 CsCl, 1 MgCl₂, 0.1 EGTA, 10 glucose, 5 HEPES, pH 7.4, adjusted with NaOH. After perfusion of β-cells for 3 min with this buffer, the first voltage step recording protocol was initiated. Subsequently, K_V and K_ATP voltage clamp recordings were performed and analysed using Clampfit software (Molecular Devices)³¹.