Direct interaction of ankyrin-G with Na_v1.5 requires two ANK repeat β-hairpin loop tips on the ankyrin-G membrane–binding domain. (A) 190-kD ankyrin-G includes a membrane-binding domain comprised of 24 consecutive ANK repeats (green), a spectrin-binding domain (black), a death domain (blue), and a C-terminal domain (red). (B) ANK repeat mutants were engineered in the context of full-length GFP 190-kD ankyrin-G and display alanine substitutions for the two residues located at the tip of each ANK repeat β-hairpin loop (red arrowheads in B and purple sites in C). (C) Crystal structure diagram of membrane-binding domain ANK repeats 13–24 (Michaely et al., 2002). Exposed charged residues on β-hairpin loop tips (sites of alanine mutagenesis) are colored in purple. (D) Relative binding (compared with wild-type GFP 190-kD ankyrin-G) of GFP 190-kD ankyrin-G ANK repeat mutants with purified Na_v1.5 DII–DIII cytoplasmic domain (n = 3; *, P < 0.05). Binding levels are corrected for the relative expression of each GFP–ankyrin-G mutant. Error bars represent SEM. (E) Na_v1.5 binding sites (arrows) superimposed on the deduced crystal structure of the ankyrin-G membrane–binding domain (ANK repeats 13–24). Ankyrin-G membrane–binding domain structure is based on the crystal structure of ANK repeats 13–24 of ankyrin-R (Michaely et al., 2002).