Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor

Abstract

Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways. This protocol has been used to study plant ion channels, including the SLAC1 channel (SLOW ANION CHANNEL-ASSOCIATED 1), in particular SLAC1 activation by either the protein kinase OST1 (OPEN STOMATA 1), Ca2+-dependent protein kinases (CPKs) or the GHR1 (GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1) transmembrane receptor-like protein. Data are presented showing reconstitution of abscisic acid activation of the SLAC1 anion channel by the 'monomeric' ABA (abscisic acid) receptor RCAR1/PYL9 (PYRABACT INRESISTANCE1 [PYR1]/PYR1-LIKE [PYL]/REGULATORYCOMPONENTS OF ABA RECEPTORS [RCAR]) by co-expressing four components of the abscisic acid signaling core. This protocol is also suitable for studying other ion channel functions and regulation mechanisms, as well as transporter proteins.