A simple in vivo method for assessing changes of membrane-bound ion channel density in Xenopus oocytes.
Heterologous expression systems, such as Xenopus oocytes, are widely used to study the regulation and the structure function relationship of ion channels and transporters. In the case of ion channels, activity can be easily measured by conventional two-electrode voltage clamping. However, this method only measures the sum of the activity of all plasma membrane-bound channels. Therefore, this measurement cannot discriminate between effects on channel density and individual channel activity. To address this shortcoming, we have developed a simple assay to detect changes of membrane-bound channel density in intact oocytes. This nonradioactive assay relies on specific antibody binding in whole live cells utilizing a simple spectrophotometric measurement. This assay is linear over a wide range of channel expression levels and provides a simple cost-effective way of monitoring changes of membrane-bound channel density. Moreover, when the heterologous proteins poorly express at the plasma membrane, this method becomes advantageous to complex biochemical cell fractionation.
Awayda
Mouhamed S
MS
Department of Physiology and Biophysics, SUNY at Buffalo, NY, USA.
Shao
Weijian
W
Vukojicic
Ivana
I
Bengrine
Abderrahmane
A
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D007473:Ion Channels / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000096:biosynthesis; D019809:Streptavidin / Q000378:metabolism; D014982:Xenopus laevis
D007473:Ion Channels; D018075:RNA, Complementary; D019809:Streptavidin