Subunit stoichiometry of heterologously expressed G-protein activated inwardly rectifying potassium channels analysed by fluorescence intensity ratio measurement

摘要

The Xenopus laevis oocyte expression system offers the unique opportunity to heterologously express many proteins simultaneously and to control the expression level for every protein individually. By using the expression of fusion constructs of variants of the green fluorescence protein (eCFP, eGFP and eYFP) with GIRK1 and GIRK4 subunits and measuring the respective fluorescence intensity ratios (FIRs) of the expressed proteins by confocal laser scan microscopy, we were able to measure the amount of each of the individual subunits expressed. At equal amounts of injected RNAs encoding GIRK1 and GIRK4, we found that approximately 2.2 GIRK4 subunits per 1 GIRK1 subunit appeared at the surface of the oocyte, suggesting the coexistence of homooligomeric GIRK4 complexes with heterooligomeric GIRK1/GIRK4 complexes. Interestingly, when the ratio of injected RNA is increased in favour of GIRK1, the subunit stoichiometry changes accordingly until, at a RNA ratio of 25:1 (GIRK1/GIRK4), the subunit stoichiometry is shifted towards a protein complex with 3:1 stoichiometry (GIRK1/GIRK4). In parallel, the amount of GIRK1 protein appearing at the surface gets greatly reduced, supporting previous studies that showed that the GIRK1 subunit needs assembly with GIRK4 for surface localization. By using a genetically encoded marker for the endoplasmic reticulum (ER), we were able to show that the subunit stoichiometry in regions of the ER, which are located directly below the plasma membrane, closely resembles that observed directly at the surface. Generally, our study reveals that the subunit stoichiometry of GIRK1/GIRK4 channels in the Xenopus laevis oocyte expression system depends to a great extent on the molar ratio of the different RNAs injected.