The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that are permeable to Na(+) and K(+). There are four members of this channel family, three of which, HCN1, HCN2, and HCN4, are expressed in the heart. Current evidence suggests that the HCN ion channel subunit family is the molecular correlate of the alpha subunit of the cardiac pacemaker current i(f). Our previous work has shown that HCN4 is the dominant isoform expressed in the rabbit sinoatrial (SA) node and that changes in tyrosine phosphorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node i(f) conductance with no change in voltage dependence. In the present study we investigate the actions of genistein, a tyrosine kinase inhibitor, on heterologously expressed HCN currents in Xenopus oocytes. Genistein had no effect on HCN1-induced currents, but reduced whole-cell currents induced by HCN2 or HCN4 and slowed activation kinetics at voltages near the midpoint of activation. In the case of HCN2 there was also a negative shift in the voltage dependence of activation that accompanies the current reduction. We have shown previously that HCN2 is the dominant isoform expressed in rat ventricular myocytes. The above results predict that genistein should reduce i(f) in the rat ventricle and cause a negative shift of voltage dependence and kinetics of activation. We tested this hypothesis by studying the effects of genistein on isolated rat ventricular myocytes. Genistein significantly reduced i(f) current density (pA/pF) (control: 12.2+/-1.8; genistein: 3.5+/-0.5; washout: 7.7+/-0.8; n=10), and caused a negative shift of the midpoint of activation by 14 mV (-133+/-1 mV for genistein and -119+/-1 mV for washout, n=7) with no change in slope factor. Our results thus suggest that i(f) in the heart and i(f)-like currents in other tissues can be regulated differentially by tyrosine phosphorylation based on isoform expression patterns.