Molecular mapping of the binding site for a blocker of hyperpolarization-activated, cyclic nucleotide-modulated pacemaker channels.

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 approximately 100-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(+) 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(+) ions observed in the KcsA crystal structure.