Sparked by our initial observation that co-expression of CNIH-2 enlarges the surface population of GluAs [22], we sought to characterize this effect in more detail. First, the amount of surface GluA1o protein was quantified in the presence and absence of CNIH-2 expression using both an extracellular epitope tagging approach and surface membrane biotinylation. In the first approach, a haemagglutinin epitope, inserted into the extracellular N-terminal domain of GluA1o, was immunostained in HeLa cells without membrane permeabilization. As shown in Figure 1A, co-expression of CNIH-2 increased the steady-state amount of GluA1o protein on the cell surface by a factor of 13.6±1.0 (n = 24; p<0.01). This effect was specific for GluA, as surface expression of the non-interacting potassium channel Kir2.1 was not affected by co-expression of CNIH-2 (data not shown). For the second experimental approach, all surface membrane proteins of HeLa cells expressing GluA1o in the presence or absence of CNIH-2 were biotinylated, affinity-purified by streptavidin-coated beads, and finally target proteins were detected and quantified by immunoblot analysis. Figure 1B shows a representative Western blot revealing a significant increase in GluA1o surface protein upon co-expression of CNIH-2. Intriguingly, also the total amount of GluA1o increased in CNIH-2 co-expressing cells.
