Characterization of heterologously expressed transporter genes by patch- and voltage-clamp methods: Application to cyclic nucleotide-dependent responses
Permanent link to this recordhttp://hdl.handle.net/10754/562965
MetadataShow full item record
AbstractThe application of patch- and voltage-clamp methods to study ion transport can be limited by many hurdles: the size of the cells to be patched and/or stabbed, the subcellular localization of the molecule of interest, and its density of expression that could be too low even in their own native environment. Functional expression of genes using recombinant DNA technology not only overcomes those hurdles but also affords additional and elegant investigations such as single-point mutation studies and subunit associations/regulations. In this chapter, we give a step-by-step description of two electrophysiological methods, patch clamp and two-electrode voltage clamp (TEVC), that are routinely used in combination with heterologous gene expression to assist researchers interested in the identification and characterization of ion transporters. We describe how to (1) obtain and maintain the cells suitable for the use with each of the above-mentioned methods (i.e., HEK-293 cells and yeast spheroplasts to use with the patch-clamp methodology and Xenopus laevis oocytes with TEVC), (2) transfect/inject them with the gene of interest, and (3) record ion transport activities. © Springer Science+Business Media New York 2013.
JournalMethods in Molecular Biology
- Functional expression of type 1 rat GABA transporter in microinjected Xenopus laevis oocytes.
- Authors: Giovannardi S, Soragna A, Magagnin S, Faravelli L
- Issue date: 2007
- Methods for studying voltage-gated sodium channels in heterologous expression systems.
- Authors: Dice MS, Kearl T, Ruben PC
- Issue date: 2006
- Identification and functional characterization of variants in human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), arising from single nucleotide polymorphisms in coding regions of the hCNT3 gene.
- Authors: Damaraju S, Zhang J, Visser F, Tackaberry T, Dufour J, Smith KM, Slugoski M, Ritzel MW, Baldwin SA, Young JD, Cass CE
- Issue date: 2005 Mar
- Fluctuations in Xenopus oocytes protein phosphorylation levels during two-electrode voltage clamp measurements.
- Authors: Cohen A, Zilberberg N
- Issue date: 2006 May 15
- Ion selectivity of pore-forming peptides and ion channels measured in Xenopus oocytes.
- Authors: Cens T, Charnet P
- Issue date: 2014