Abstract
Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) channels are similar in structure and function to voltage-gated potassium channels. Sequence similarity and functional analyses suggest that the HCN pore is potassium channel-like, consisting of a selectivity filter and an activation gate at the outer and inner ends, respectively. In GYG-containing potassium channels, the selectivity filter sequence is 'T/S-V/I/L/T-GYG', forming a row of four binding sites through which potassium ions flow. In HCNs, the equivalent residues are 'C-I-GYG', but whether they also form four cation binding sites is not known. Here, we focus on the anomalous filter residue of HCNs, the cysteine located at the inner side of the selectivity filter. In potassium channels, this position is occupied by threonine or serine and forms the fourth and most internal ion binding site of the selectivity filter. We find that this cysteine in HCNs does not contribute to permeation or form a fourth binding site.