We first examined the whole cell conductance of the cells transfected using the SV40 and the CMV promoters (Fig. 2). Expectedly, 24 h after transfection, the whole cell conductance of SV40 plasmid cells was significantly lower than that of CMV promoter. Interestingly, selleck compound 48 h after transfection, the whole cell conductance was comparable between high and low expression cells. If the abilities
of these promoters did not change over time, this result suggests that the half-lives of Kir2.1 were different depending on the expression level. We next attempted to measure the half-life. We pulse-labeled the SNAP-Kir2.1 with a membrane-permeable fluorescent substrate for the SNAP tag, SNAP-cell-TMR-Star, 24 h after the transfection. SNAP-cell-TMR-Star covalently binds to the SNAP tag domain (Fig. 1A). After the washing-out of unbound dye for 2 h, we examined it microscopically and found that the SNAP-Kir2.1 fusion protein was successfully labeled in both cells transfected using the SV40 and the CMV promoters (Fig. 3A). The fluorescence of the cells transfected with the CMV promoter plasmid was significantly higher than that of the cells transfected with the SV40 promoter plasmid as we observed in whole cell current. Reportedly, HEK293 cells
endogenously express the O6-alkylguanine-DNA-alkyltransferase PF-562271 (Keppler et al., 2004), but the background fluorescence was negligible compared with SNAP-Kir2.1 (data not shown). This is probably due to the high level expression of SNAP-Kir2.1 and the 20-fold higher activity of the mutant SNAP-tag, which we used here. Initially, the fluorescence was mostly located at the plasma membrane of 293T cells in both cases, but some intracellular, punctuated fluorescence was observed in the CMV promoter-transfected cells (Fig. 3A). The intensity of the fluorescence decreased over
time. In the high-expression cells transfected with the CMV promoter plasmid, most SNAP-Kir2.1 proteins were internalized from the plasma membrane and the fluorescence was punctuated 24 h after labeling. In the low-expression cells MTMR9 transfected with the SV40 promoter plasmid, most SNAP-Kir2.1 proteins were still located at the plasma membrane 24 h after the labeling, and some even after 48 h. We measured the fluorescence in the whole area of each cell and estimated the half-lives of the SNAP-Kir2.1 protein expressed by the two promoters (Fig. 3B). The fluorescence decreased faster in the high-expression cells than low-expression cells. The half-life was significantly shorter in the high-expression cells (18.2±1.9 h) than in the low-expression cells (35.1±2.3 h, n=5, p<0.0005, Student′s t-test) ( Fig. 3C). This result supports a hypothesis that a high level of Kir2.1 accelerates its own degradation. Microscopic measurement of fluorescence intensity can be affected by cell division, i.e., the density of labeled SNAP-Kir2.