Concatenation of Kv1.X genes, expression in HEK-293 cells, and detection of complete heterotetrameric K⁺ channels on the plasmalemma. (A) Representation of the attachment to Kv1 genes of half-linkers and restriction sites at the 5′ (black) and 3′ (gray) end of each gene for assembly of concatemers in pIRES2-EGFP. Complete linkers form during the assembly of concatemers retaining all the sequences in the same open reading frame. (B) Adjacent and diagonal arrangements of the two α-subunit genes in the tandem-linked constructs, named in a clockwise direction (forward and reverse), used to produce the concatenated tetrameric proteins composed of Kv1.1 (green) and Kv1.2 (brown), based on a PDB file of the structure of the Kv1.2 α/β–subunit complex (Long et al., 2005). (C) HEK-293 cells transfected with pIRES2-EGFP containing the following constructs are shown in lanes: 1, Kv1.2; 2, Kv1.2-1.2; 3 and 4, Kv1.1-1.1-1.2-1.2; 5, Kv1.1-1.2-1.1-1.2; 6, Kv1.2-1.2-1.1-1.1; 7, Kv1.2-1.1-1.2-1.1. After expression for 48 h, the cells were lysed and subjected to SDS-PAGE (on 4–12% polyacrylamide gels), followed by Western blotting with the antibodies specified: Kv1.2 (lanes 1–3 and 5–7) and Kv1.1 (lane 4). (D) Intact cells expressing tetramers (as in C) were washed, biotinylated, detergent solubilized, precipitated with streptavidin-agarose beads, and analyzed using antibodies specific for Kv1.2 or Kv1.1. Lanes: 1 and 5, Kv1.1-1.1-1.2-1.2; 2 and 6, Kv1.1-1.2-1.1-1.2; 3 and 7, Kv1.2-1.2-1.1-1.1; 4 and 8, Kv1.2-1.1-1.2-1.1. Mobilities of standard proteins are indicated. Cells subjected to a mock transfection with the vector lacking any K⁺ channel construct, and biotinylated as before, did not show any band reactive with Kv1.1 or 1.2 IgGs. Exclusive extracellular labeling of intact HEK-293 cells by the biotinylation procedure was established from the inability to biotinylate actin, an intracellular protein, unless a cell lysate was used.