Voltage-clamp recording with a single Xenopus oocyte for INX-6 hemichannels
This protocol is extracted from research article:
Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids
Sci Adv, Feb 12, 2020; DOI: 10.1126/sciadv.aax3157

A series of N-terminal deletion mutants (ΔN2-3, ΔN2-6, and ΔN2-19) of INX-6 was generated according to the PrimeSTAR Mutagenesis Basal Kit protocol (Takara). Complementary DNAs (cDNAs) for INX-6 and its mutants were cloned into pGEM-HeFx plasmids (68). The plasmids were linearized using restriction enzymes and then applied to an RNA preparation kit (T7 mMESSAGE mMACHINE; Ambion) according to the manufacturer’s protocol. An adult Xenopus laevis female was anesthetized with MS-222 (Merck), and the ovarian lobes were collected using a surgical knife and forceps. The eggs were treated with collagenase solution (1 mg ml−1 collagenase type I, Life Technologies) in OR2 buffer (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM Hepes, adjusted to pH 7.5 using NaOH) at 18°C for 1.5 hours. Stage V and VI oocytes were collected manually and used for cRNA injection. cRNA (10 ng) was coinjected with antisense oligonucleotide DNA for Xenopus cx38 (10 ng) into Xenopus oocytes. For the negative control, only antisense oligonucleotide was injected. Oocytes injected with cRNA were incubated for 3 days at 18°C in ND96 buffer (93.5 mM NaCl, 2 mM KCl, 1.8 mM MgCl2, 2 mM MgCl2, and 5 mM Hepes, adjusted to pH 7.5 using NaOH). Hemichannel currents were recorded from a single oocyte using Multi-Electrode Clamp Amplifier iTEV90. Current and voltage electrodes were prepared with a micropipette puller (P-1000, Sutter Instrument) to obtain a resistance of 0.5 to 1.0 megohm when filled with an internal solution containing 3 M KCl, 10 mM ethylene glycol tetraacetic acid (EGTA), and 10 mM Hepes, adjusted to pH 7.4 using KOH. Modified ND96 solution without CaCl2 was used as the bath solution. To obtain the hemichannel current, the cells were initially clamped at −40 mV and then subjected to 5-s voltage steps from −30 to +50 mV in 10-mV increments.

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