Schematic of the structure of spHCN channel subunits. (A) Schematic of the putative topology of an spHCN subunit. Locations of the charged S4 voltage sensor, the S4–S5 linker, the post-S6/C-linker, the CNBD, and the binding site for cyclic nucleotide monophosphate (cNMP) are shown. (B) Sequence alignments of the S4, S4–S5 linker, pore-lining S6, and post-S6/C-linker regions of spHCN1 (GenBank accession no. CAA76493), with human Kv1.2 (NCBI Protein database accession no. NP_004965), rat Kv1.2 (NCBI Protein database accession no. NP_037102), and KcsA (UniProtKB/Swiss-Prot sequence P0A334) channel subunits. Alignments were made using ClustalW2. Mutations of spHCN1 discussed in Results and Discussion are shown as bold and underlined.