title: The Xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry.
abstract: The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Rudokas
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Varga
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis.
authors.forename: Angela R
authors.initials: AR
authors.lastname: Schubert
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis.
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Asaro
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis; jonsilva@wustl.edu.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Silva
mesh_terms: D000818:Animals; D005260:Female; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000096:biosynthesis / Q000235:genetics; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D014982:Xenopus laevis
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel