Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes.
The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (approximately 1mm diameter), (b) it has an established capacity to produce-from microinjected mRNAs or cRNAs-exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion transporters as green fluorescent protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl(-), H(+) (and hence base equivalents like OH(-) and HCO(3)(-)), K(+), and Na(+) is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates.
PubMed ID: 20051266
PMC ID: PMC2905798
Article link: Methods.
Grant support: R01 NS018400-20 NINDS NIH HHS , R37 DK030344-27 NIDDK NIH HHS