If (or Ih), encoded by the hyperpolarization-activated, cyclic nucleotide-gated (HCN1-4) channel gene family, contributes significantly to cardiac pacing. Bradycardic agents such as ZD7288 that target HCN channels have been developed, but the molecular configuration of their receptor is poorly defined. Here, we probed the drug receptor by systematically introducing alanine scanning substitutions into the selectivity filter (C347A, I348A, G349A, Y350A, G351A in the P-loop), outer (P355A, V356A, S357A, M358A in the P-S6 linker), and inner (M377A, F378A, V379A in S6) pore vestibules of HCN1 channels. When heterologously expressed in human embryonic kidney 293 cells for patch-clamp recordings, I348A, G349A, Y350A, G351A, P355A, and V356A did not produce measurable currents. The half-blocking concentration (IC50) of wild type (WT) for ZD7288 was 25.8+/-9.7microM. While the IC50 of M358A was identical to WT, those of C347A, S357A, F378A, and V379A markedly increased to 137.6+/-56.4, 113.3+/-34.1, 587.1+/-167.5, and 1726.3+/-673.4microM, respectively (p<0.05). Despite the proximity of the S6 residues studied, M377A was hypersensitive (IC50=5.1+/-0.7microM; p<0.05) implicating site specificity. To explore the energetic interactions among the S6 residues, double and triple substitutions (M377A/F378A, M377A/V379A, F378A/V379A, and M377A/F378A/V379A) were generated for thermodynamic cycle analysis. Specific interactions with coupling energies (deltadeltaG)>1kT for M377-F378 and F378-V379 but not M377-V379 were identified. Based on these new data and others, we proposed a refined drug-blocking model that may lead to improved antiarrhythmics and bioartificial pacemaker designs.