Cardiac pacemaker current, if, is generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Our previous studies demonstrated that altered tyrosine phosphorylation can modulate the properties of both if and HCN channels. To assess a hypothesis that the intracellular tyrosine kinase Src may play a role in modulation by tyrosine phosphorylation of if, we cotransfected HEK293 cells with HCN4 and Src proteins. When HCN4 was cotransfected with a constitutively activated Src protein (Src529), the resultant voltage-dependent HCN4 activation was positively shifted (HCN4: V1/2=−93 mV; Src529: V1/2=−80 mV). The activation kinetics were accelerated at some potentials but not over the entire voltage range tested (eg, at −95 mV, τ_act(HCN4)=3243 ms; τ_act(Src529)=1113 ms). When HCN4 was cotransfected with a dominant negative Src protein (Src296), the HCN4 activation was shifted more negative to a smaller degree (HCN4: V1/2=−93 mV; Src296: V1/2=−98 mV; statistically insignificant) and the activation kinetics were slowed at most test potentials (eg, at −95 mV, τ_act(Src296)=7396 ms). Neither Src529 nor Src296 significantly altered HCN4 current density. Coimmunoprecipitation experiments revealed that Src forms a complex with HCN4 in HEK293 cells and in rat ventricular myocytes. Our data provide a novel mechanism of if regulation by Src tyrosine phosphorylation.
*New York College of Osteopathic Medicine of the New York Institute of Technology, New York
†Institute of Molecular Cardiology and Department of Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook
‡Center for Interdisciplinary Research in Cardiovascular Sciences, West Virginia University, Morgantown, WV
Reprints: Han-Gang Yu, West Virginia University, Morgantown, WV 26506 (e-mail: email@example.com).
Received for publication December 4, 2005; accepted March 14, 2006
Supported by a grant from American Heart Association (National Scientist Development Grant, 0030124N, HGY) and HL075023 from NHLBI (HGY) and was presented in abstract form at the meeting of the Biophysical Society in February 2005. I.S.C. is supported by HL28958 and HL67101 from the National Heart, Lung, and Blood Institute.