Flaming brown horizontal electrode puller

Two‐electrode voltage clamp electrophysiology was used to record currents produced by activation of the expressed Asu‐ACR‐16 receptor (Buxton et al., 2014). Four hours prior to recording, 100 μM BAPTA‐AM (final concentration), a cell‐permeant calcium chelator, was added to the oocyte incubation solution to prevent activation of endogenous calcium‐activated chloride channels during recordings. Recordings from non‐injected oocytes served as control experiments. Recordings were made using an Axoclamp 2B amplifier (Warner Instruments, Hamden, CT, USA) with the oocytes voltage clamped at −60 mV and data acquired on a computer with Clampex 9.2 (Molecular Devices, Sunnyvale, CA, USA). The microelectrodes used to impale the oocytes were pulled using a Flaming/Brown horizontal electrode puller (Model P‐97; Sutter Instruments, Novato, CA, USA) set to pull micropipettes that when filled with 3 M KCl had a resistance of 20–30 MΩ. The micropipettes tips were carefully broken with a piece of tissue paper in order to achieve a resistance of 2–5 MΩ in recording solution (100 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂.2H₂O and 5 mM HEPES, pH 7.3). The low resistance pipettes allowed large currents to be passed to maintain adequate voltage clamp.