Two-Electrode Voltage-Clamp Electrophysiology.

Kir3.1/Kir3.2 channel currents were recorded from oocytes using the two-electrode voltage-clamp technique.⁴⁶ Oocytes were initially superfused with ND98 solution (in mM): 98 NaCl, 1 MgCl₂, and 5 HEPES at pH 7.5 (NaOH). Glass electrodes having tip resistances of 0.8–1.0 MΩ were used to voltage-clamp oocytes at a holding membrane potential of −40 mV (GeneClamp 500B, Axon Instruments). A voltage protocol was evoked every 1 or 5 s and consisted of a step change to −80 mV (50 ms in duration), followed by a ramp from −80 to +20 mV (200 ms in duration).

After establishing baseline currents during the first few minutes of recording, the bath solution was changed to a “high K⁺ solution” that consisted of (in mM) 20 KCl, 78 NaCl, 1 MgCl₂, and 5 HEPES at pH 7.5 (NaOH). Large inwardly rectifying K⁺ currents were associated with the transition to high K⁺ solution attributed predominantly to constitutively active “basal” Kir3.1/Kir3.2a channel currents. Application and washout of ACh and the various TPN_Q peptide concentrations in the 20 mM K⁺ solution were performed by a perfusion barrel positioned next to the recorded oocyte for rapid solution exchange.⁴⁶ All recordings were performed at room temperature (21–23 °C). The membrane currents were digitized using an A/D acquisition board (Digidata 1440 acquisition system, Axon Instruments) and then later analyzed using pCLAMP software (version 10.6).