4.5. Two-Electrode Voltage Clamp Recording from Xenopus Oocytes

Macroscopic mKir4.1 and rKir1.1 channel currents were recorded by two-electrode voltage clamp recording techniques where oocytes were initially superfused with the following bath 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 clamp oocytes at a holding membrane potential of −80 mV. After establishing a baseline holding current, the bath solution was changed to a “high K⁺ solution” that was comprised of an equal molar substitution of NaCl for KCl. For mKir4.1 recordings, the extracellular K⁺ concentration was 98 mM, and for rKir1.1 recordings, the concentration was 20 mM K⁺. These concentrations were determined empirically to control for peak K⁺ current amplitudes, and were attributed to differences in Kir4.1 vs Kir1.1 channel expression, single-channel conductance, and single-channel open time probability. From the −80 mV holding potential, large inward K⁺ currents were evoked and were due to the expressed Kir channels, as un-injected oocytes yielded inward currents of <100 nA (data not shown).

Rapid application and washout of 100 nM TPN_Q or 1 mM BaCl₂ (dissolved in high K⁺ solutions) was performed as described previously [40]. TPN_Q (lyophilized solid, Tocris Bioscience, Bristol, UK) was initially dissolved in water as a 100 μM stock solution, then aliquoted and stored at −23 °C until used on the day of the experiment by diluting in the high K⁺ electrophysiological recording solution. Voltage ramps from −80 to +20 mV (200 ms in duration) were evoked periodically to assess the inward rectification characteristics of Kir channel currents during changes in the recording solutions. All recordings were performed at room temperature (21–23 °C). The membrane currents were digitized, stored, and later analyzed using an Analog-to-Digital acquisition board and PC computer (pCLAMP software, Digidata 1200 acquisition system, Axon Instruments, Foster City, CA, USA). Experiments were replicated in 7 oocytes.