Fig 2: Kv11.1 α-subunit schematic and sequence of channel mutation.A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

Fig 3: Comparison of experimental and computational analysis of amiodarone block. (A) Low energy score structure of amiodarone (wheat coloured space filling representation) docked into the MthK-based hERG model extended to include the S5 helix. One subunit of the model is rendered as a Connelly surface coloured as a heat map according to amino acids whose mutation to Ala attenuates drug binding as defined in panel (B). K+ ions in the S1 and S3 positions of the selectivity filter are pink spheres. (B) One subunit of the hERG model extracted from panel (A) and coloured to define residues whose Ala mutation attenuates amiodarone block by: 17–22-fold (deep red); approx. 10-fold (pale red); 5–7-fold (mauve); the latter group comprised T623 and G648 (Table 2), however G648 lies behind Y652 and is hidden in this view. (C) The same subunit coloured according to residues that make interactions (as defined in [32]) with amiodarone in low energy docked states. Annotations in panel C define residues that make direct interactions with drug in docking and whose mutation to Ala attenuates drug block (except for A653 which was not mutated experimentally). Annotations in panel (B) define residues whose Ala mutation attenuates amiodarone block but which do not make direct interaction with drug in low energy score docked states. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 4: Nedd4-2-mediated ubiquitination of Kv11.1 was increased in pCH. (A) Representative immunoblots showing Nedd4-2, phosphorylated Nedd4-2 (p-Nedd4-2) and Rab11 proteins and corresponding quantifications of band densities in myocardium from control (CON) (n = 5) and pCH (Ang II, n = 7). GADPH was used as an internal control to normalize the bands. (B) Representative blots immunoprecipitated (IP) by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-Nedd4-2 antibody. Quantification of band densities was shown as the ratio of Nedd4-2 to Kv11.1. (C) Representative blots immunoprecipitated by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-ubiquitin antibody. Quantification of band densities was shown as the ratio of ubiquitin to Kv11.1. (D) Proteins from control and pCH myocardium were immunoprecipitated with an anti-Kv7.1 or anti-IgG antibody, and IB was performed by using anti-Nedd4-2 antibody. IgG was used as negative controls. n = 3–4. *p < 0.05 and ***p < 0.001 versus CON. ns: not statistically significant.

Fig 5: Knock down of hERG channel protein mimics pro-apoptotic effects of doxazosin.(A–C) Representative microphotographs reflect viability of LNT-229 cells treated with small interfering RNA (siRNA) to suppress hERG expression (C) compared to controls exposed to DMSO (solvent; A) or scrambled siRNA (B). Scale bar, 100 µm. (D) XTT assay-based quantification of apoptotic cells following indicated treatment normalized to solvent controls (n = 4 to 6 assays;). Reduced viability associated with doxazosin or hERG siRNA treatment was further increased by simultaneous application of both compounds. (E, F) Western blot analysis (E) and corresponding mean hERG expression levels normalized to GAPDH expression (F) confirmed hERG protein knock down by ~58%. Data are given as mean ± SEM (**p<0.01; ***p<0.001). Scr, srambled siRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.