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. 2010 Nov 3;99(9):2853–2862. doi: 10.1016/j.bpj.2010.08.049

Figure 1.

Figure 1

Specific labeling of functional BKCa channels expressed in COS-7 cells using QDs. (A) Schematic illustration of the rat BKCa channel α-subunit (or rSlo) used in this experiment. An acceptor peptide (AP) was tagged at the N-terminus of rSlo, and red fluorescent protein (RFP) was tagged at the C-terminus. (B) Specific labeling of AP-rSlo-RFP channels using QDs. COS-7 cells were transfected with BirA-ER and RFP (first row), AP-rSlo-RFP (second row), or AP-rSlo-RFP with BirA-ER (third row). In the fourth row, magnified views (3×) of the highlighted regions in the third row are shown. In the case of BirA-ER shown in the first row, a plasmid harboring RFP (pTag-RFP-C1; Evrogen, Moscow, Russia) was cotransfected in order to visualize the transfectants. Cells were labeled with streptavidin-conjugated QD605 (Invitrogen). Cells expressing either RFP or AP-rSlo-RFP are red (left column) and QD605 is blue (center column). Merged images are also shown (right column). Scale bar, 10 μm. (C) Electrophysiological characterization of rSlo, AP-rSlo-RFP, and QD-labeled AP-rSlo-RFP channels (QD:AP-rSlo-RFP). Representative current traces of rSlo, AP-rSlo-RFP, and QD-labeled AP-rSlo-RFP channels (QD:AP-rSlo-RFP) are shown. Ionic currents were evoked with 100-ms voltage steps from the holding potential of –100 mV to test potentials of 20, 50, and 80 mV. Intracellular Ca2+ concentration was buffered at 10 μM. The response of untransfected HEK293 cells to voltage pulses is also shown as a control.