Ivabradine is a molecule that selectively blocks the sinoatrial pacemaker “f” channels acting specifically from the intracellular site (1), and exclusively decreases heart rate without undesired cardiovascular side effects. Specifically, ivabradine has no negative inotropic or lusitropic effects and preserves ventricular contractility, and is therefore a primary tool in the pharmacological treatment of chronic stable angina (2). Native f-channels are composed of HCN subunits, of which 4 isoforms are known. Experimental evidence indicates that f-channels are tetramers composed mostly of HCN4 subunits with a possible minor contribution of HCN1. The mechanisms of ivabradine block of sinoatrial f channels and HCN4 and HCN1 isoforms have been investigated in our laboratory (3,4). The properties of native and HCN4 channels block are similar, in that both display use- and current-dependence. The block of ivabradine of HCN1 channels, on the other hand, is still use-dependent but the current flow does not affect drug-channel interaction. Data presented in this poster were collected with the aim of identifying critical residues in the pore-lining region involved in binding of ivabradine to human HCN4 channels. We focused our attention to two aminoacids (Y506 and I510) that are homologous to those involved in the binding of another I_f inhibitor compound (ZD7288) to HCN1 channel(5). We proceeded by individually substituting the two aminoacids by alanine residues (Y506A and I510A) and transfecting the mutated hHCN4 cDNAs into HEK293 cells. Electrophysiological studies were then performed in the whole-cell configuration at 32°C. We initially characterized the block with 30 µM drug by applying an activation/deactivation protocol (-140/+5 mV, 0.5 Hz) from a holding potential of -35 mV. Mean % blocks ± sem were 87.1±3.2 (n=5), 31.9±2.5 (n=4), and 41.2±6.0 (n=4) for wt, Y506A, and I510A channels, respectively. Statistical comparison revealed a decreased drug affinity for both mutations relative to wt channels (p<0.05, t-test). A full dose response was obtained for wt and Y506A channels; fitting with the Hill relation yielded Kd values of 1.6 µM and 56.1 µM and Hill slopes of 0.72 and 1.2, respectively; the two curves were significantly different (p0.05, t-test), suggesting that the access of the drug to its binding site is not affected by the mutations. In conclusion, these data allow a preliminary identification of residues of the HCN4 channel that likely contribute to the binding of ivabradine and channel block.