Molecular Mapping of the Binding Site for a Blocker of Hyperpolarization-Activated, Cyclic Nucleotide-Modulated Pacemaker Channels

Abstract

Hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels mediate rhythmic electrical activity of neural and cardiac pacemaker cells. Drugs that block these channels slow the beating rate of the heart and are used to treat angina. Here, we characterized the effect of the HCN channel blocker, ZD7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] on HCN2 channels that were heterologously expressed in Xenopus oocytes. A site-directed mutagenesis approach was used to identify specific residues of the mouse HCN2 channel pore that interact with ZD7288. Two residues (Ala425 and Ile432) located in the S6 transmembrane domain were found to be the primary determinants for block of HCN2 channels by ZD7288. I432A mutant HCN2 channels were ϳ100-fold less sensitive to block by ZD7288. Substitution of Ile432 with more hydrophobic residues (Phe, Leu, or Val) caused only modest shifts in the IC 50 for the drug. HCN1 channels have a Val (Val390) in the equivalent position of Ile432 and are less sensitive to block by ZD7288. Accordingly, mutation of this Val390 to Ile in HCN1 increased the sensitivity of these channels to drug block. Mutation of Ala425 and Ile432 also attenuated the block of HCN2 by the more potent blocker cilobradine. An HCN2 homology model based on the bacterial KcsA K ϩ channel predicts that the phenyl ring of ZD7288 occupies a hydrophobic cavity formed by Ala425 and Ile432 and that the charged ring aligns with the axis of the inner pore closely corresponding to the localization of K ϩ ions observed in the KcsA crystal structure.Pacemaker channels are activated by membrane hyperpolarization and conduct an inward cation current that contributes to spontaneous activity of specialized pacemaker cells in the heart (Baruscotti et al., 2005) and synaptic integration and network rhythmicity of central neurons (Biel et al

Nicotine Blocks the Hyperpolarization-Activated CurrentIhand Severely Impairs the Oscillatory Behavior of Oriens-Lacunosum Moleculare Interneurons

“…Our modeling suggests that the bending amplitude of the S6 helix during gating is significantly smaller than that found in MthK channels (Jiang et al, 2002). The model has been further validated by a recent study (Cheng et al, 2007), in which the amino acids involved in the interaction between ZD 7288 and cilobradine with HCN channels has been identified. Docking procedure.…”

“…However, this hypothesis should be validated with point-directed mutagenesis experiments. Interestingly, the poses of the ligands are highly similar to those described recently for ZD 7288 and cilobradine (Cheng et al, 2007).…”

Tanshinone IIA Selectively Enhances Hyperpolarization-Activated Cyclic Nucleotide–Modulated (HCN) Channel Instantaneous Current

“…1C) (18 -20). Similar V 1/2 , k, and min-Po values were found when compared to those reported in the literature (20,21).…”

“…Figure 1C showed that in addition to the blockade of the voltage-dependent current, ZD7288 (one of the HCN-channel blockers) also blocked the inward instantaneous current at hyperpolarized potentials. Alanine mutagenesis results suggested that ZD7288 binds in the pore of either HCN2 or HCN1 channels (20) from the intracellular side of the membrane (23). Different from ZD7288, tanshinone IIA selectively enhanced instantaneous current.…”

The role of an ancestral hyperpolarization activated cyclic nucleotide-gated K+-channel in branchial acid-base regulation in the green crab,Carcinus maenas(L.)

“…1). The major binding sites for ZD7288 were identified as Ala-425 and Leu-432, as recently described by Cheng et al (2007).…”