title: Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA.
abstract: The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.
authors.affiliation: a Department of Physiology & Biophysics ; The Chicago Medical School; Rosalind Franklin University ; Chicago , IL USA.
authors.forename: Henry
authors.initials: H
authors.lastname: Sackin
authors.forename: Mikheil
authors.initials: M
authors.lastname: Nanazashvili
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Makino
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger
keywords: ROMK; inward rectifier; patch-clamp; proteoliposome; renal