Whole‐cell patch‐clamp electrophysiology

Currents were recorded using the whole‐cell patch clamp in a voltage clamp configuration in tsA201 cells transiently transfected with the channel of interest. The cover slip with the cells was placed in a recording chamber filled with an external medium composed of 145 mM NaCl, 2.5 mM KCl, 3 mM MgCl₂, 1 mM CaCl₂ and 10 mM HEPES (pH to 7.4, using NaOH). In experiments where external [K] was varied, NaCl was replaced with KCl to give the appropriate concentration of K. The internal medium used in the glass pipette comprised 150 mM KCl, 3 mM MgCl₂, 5 mM (or 0.1 mM) EGTA and 10 mM HEPES (pH to 7.4, using KOH). Modulatory compounds were applied by bath perfusion at a rate of 4–5 mL·min⁻¹. Complete exchange of bath solution usually occurred within 100–120 s; as such, any effect of these compounds on current amplitude was only measured once the response had reached steady state.

All the data presented have been collected at room temperature (19–22°C). The transfected cells were detected using a fluorescent microscope with UV light. Cells were voltage clamped using an Axopatch 1D or Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) and low‐pass filtered at 5 kHz before sampling (2–10 kHz) and online capture.

In order to study the potassium leak current, a ‘step‐ramp’ voltage protocol was used. For the step component of the protocol, cells were hyperpolarized from a holding voltage of −60 to −80 mV for 100 ms then stepped to −40 mV for 500 ms. For the ramp, cells were then stepped to −120 mV for 100 ms, followed by a 500 ms voltage ramp to +20 mV and a step back to −80 mV for another 100 ms, before being returned to the holding voltage of −60 mV. This protocol was composed of sweeps that lasted 1.5 s (including sampling at the holding voltage) and was repeated once every 5 s (see also Veale et al., 2014b).