Established Protocols for cRNA Expression and Voltage-Clamp Characterization of the P2X7 Receptor in Xenopus laevis Oocytes

AbstractP2X7 receptors (P2X7Rs) are fast ATP4−-gated ion channels that, like other members of the P2X receptor family, function as homotrimers. A high-resolution cryo-EM structure of the full-length rat P2X7R is available. Using voltage-clamp experiments in Xenopus laevis oocytes, even the earliest steps of P2X7R activation can be quantitatively recorded in the millisecond range. Site-directed mutagenesis combined with voltage-clamp recordings can reveal residues and domains of the P2X7R involved in ATP4− binding, gating (i.e., opening and closing of the channel pore) and ion selectivity. We present here proven voltage-clamp protocols that take into account requirements that are important at the levels of cDNA and vector sequences, cRNA synthesis, and Xenopus laevis oocyte isolation for reliable results.Key wordsProtein expression in Xenopus laeviscRNA synthesisPolyadenylationTwo-microelectrode voltage clampPatch clampU-tube