Native sinoatrial f-channels play a key role in the generation of the spontaneous electrical activity of the heart. According to this role, the inhibition of the I_f current by heart rate-reducing agents causes a selective reduction of the heart rate. f-channels are composed of HCN subunits, of which 4 isoforms are known; studies have identified residues of the pore regions of HCN1 and HCN2 isoforms that are critical for the binding of two members of the heart rate-reducing family: ZD7288 and cilobradine. We have identified the corresponding residues in HCN4, the main isoform expressed in the sinoatrial region, and replaced these residues by alanine (or valine), since these substitutions are normally used for investigating the role of specific residues in drug-binding mechanisms. WT and mutated channels have been heterologously expressed in HEK293 cells in order to verify by patch-clamp experiments if these mutations alter the biophysical properties of HCN4 channels. We found that the mutations A503V and Y506A induce a leftward shift of the activation curve and slow down the activation kinetics. The I510A and M508A mutations did not affect the activation curve but they did modify the activation kinetics: the I510A mutation clearly speeded up while the M508A slowed down the channel activation. This information will be useful for investigating a potential role of these residues in the binding of heart rate-reducing agents to the HCN4 channel.