Pflugers Arch - Eur J Physiol (2004) 447: 392–400 DOI 10.1007/s00424-003-1204-y CELL AND MOLECULAR PHYSIOLOGY Han-Gang Yu . Zhongju Lu . Zongming Pan . Ira S. Cohen Tyrosine kinase inhibition differentially regulates heterologously expressed HCN channels Received: 4 September 2003 / Revised: 10 October 2003 / Accepted: 15 October 2003 / Published online: 21 November 2003 # Springer-Verlag 2003 Abstract 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 phos- phorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node i f conduc- tance 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. Keywords i f . HCN channels . Tyrosine phosphorylation . Genistein . Rat ventricular myocytes Introduction Phosphorylation is an important regulatory mechanism governing many cell activities. Ion channels are no exceptions, since the gating and ion permeation of calcium channels, sodium channels and potassium channels have been reported to change with phosphorylation state [3]. Although the majority of previous studies have focused on serine-threonine kinases, a growing body of literature also has demonstrated important effects on ion channels mediated through changes in tyrosine phosphorylation state [3]. Our own previous work on the cardiac pacemaker current, i f , (f for “funny”, since the current is activated by hyperpolarization, as opposed to most ionic currents activated by depolarization) has demonstrated that sinoatrial (SA) node i f is reduced by inhibition of tyrosine kinase activity by genistein or herbimycin A and enhanced by its stimulation with epidermal growth factor (EGF), which also increases heart rate [12, 13, 16, 17]. Within the last 5 years a family of four genes encoding the hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN) have been cloned and expressed in heterologous expression systems [5, 10, 11, 14]. Three members of this channel family are expressed non- uniformly in cardiac tissues [15]: abundant HCN4 and low levels of HCN2 transcripts are expressed in the SA node whilst, in contrast, much higher levels of HCN2 than HCN4 transcripts are present in the ventricle. HCN1 transcripts are expressed at low levels in the SA node and largely absent in ventricle. This differential expression H.-G. Yu (*) New York College of Osteopathic Medicine of New York Institute of Technology, New York, N.Y., USA e-mail: hgyu@nyit.edu Tel.: +1-516-6863811 Fax: +1-516-6863832 Z. Lu . Z. Pan . I. S. Cohen Institute of Molecular Cardiology and Department of Physiology and Biophysics, SUNY at Stony Brook, Stony Brook, N.Y., USA