Figure 4
From: Intracellular hemin is a potent inhibitor of the voltage-gated potassium channel Kv10.1
Involvement of the C-linker motif CxHx8H for the impact of hemin on Kv10.1. (A) Two opposing subunits of rat Kv10.1 (5K7L)53 indicating for one subunit (green) the transmembrane segments with the voltage sensor, the N-terminal PAS domain (cyan) and the deletion site G190 to yield Δ2-190, as well as the C-terminal C linker (magenta) which ends in the identified hemin-binding motif encompassing C541..H552 (red). For clarity, the other two subunits in the view direction as well as the calmodulin molecules present in 5K7L are not shown. (B) Multiple sequence alignment of the C-linker region of the CNBD family of channels shows that the heme-binding motif (CxHx8H) is conserved in some members of the EAG family (Kv10.1, Kv11.1), while absent in ELK1 (Kv12.1), in the plant K+ channels AKT1 and KAT1, and the cyclic nucleotide-gated channels HCN1 and CNGA2. The putative heme-binding motif is highlighted. The vertical blue lines mark the peptide sequence used for the synthesis of 22-mer peptides. (C) Representative inside-out current traces from Xenopus oocyte membrane patches at 40 mV before (black, blue) and after hemin application (100 nM in 1 mM reduced GSH, red) for Kv10.1 wild type (wt) and variant C541A:H543A:H552V (CA:HA:HV). (D) Time course of current reduction upon application of 100 nM hemin in reduced GSH for the indicated channel types. Data are mean ± sem with n in parentheses (for wt and CA:HA:HV sem in shading). Straight lines connect data points for clarity.
