Aims: 

Heterologous expression in oocytes of Xenopus laevis allows the controlled simultaneous expression of up to >10 different proteins. Little data exist, so far, on the exact quantitation of the protein stochiometry of expressed excitability proteins. We studied the exact protein stochiometry between the GIRK1 and GIRK4 subunits of I_K+ACh at different ratios of injected RNA.

Methods: 

N-terminal fusion proteins between the GIRK1 and GIRK4 subunits and variants of the green fluorescence protein (eGFP, eCFP, eYFP) were engineered and expressed in Xenopus laevis oocytes at different RNA ratio's. Using confocal miscroscopy, the subunit stochiometry was analysed by the Fluorescence Intensity Ratio (FIR) method.

Results: 

It was found, that the subunit stochiometry GIRK1:GIRK4 was 0.46 ± 0.02 (N = 9 different batches of oocytes) at low magnification, using the 20× objective, when equal amounts of RNA were injected (10 ng per oocyte for each RNA species). When the ratio of injected GIRK1 RNA to GIRK4 RNA is raised to 5:1 and 25:1 the protein subunit stochiometry GIRK1:GIRK4 changes to 1.46 ± 0.13 (N = 9) and 2.82 ± 0.38 (N = 9), respectively. When oocytes were viewed at high magnification and a fluorescence marker for the endoplasmic reticulum (ER) was used, it could be shown that the subunit stochiometry is identical in the plasmamembrane and in the ER.

Conclusion: 

These results demonstrate the coexistence of GIRK1/ GIRK4 heterooligomeric as well as of GIRK4 homooligomeric complexes in the plasmamembrane upon injection of equal RNA amounts encoding both subunits. The GIRK1:GIRK4 stochiometry can be shifted towards a 3:1 ratio, by injecting GIRK1 RNA in excess.