+ K Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current If Guido Michels1, Fikret Er1, Ismail F. Khan1, Jeannette Endres-Becker1, Mathias C. Brandt1, Natig Gassanov1, David C. Johns2, Uta C. Hoppe1,3* 1 Department of Internal Medicine III, University of Cologne, Cologne, Germany, 2 Gene Vector Center, The Johns Hopkins University, Baltimore, Maryland, United States of America, 3 Center for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany Hyperpolarization-activated, cyclic nucleotide sensitive (HCN) channels underlie the pacemaker current If, which plays an essential role in spontaneous cardiac activity. HCN channel subunits (HCN1-4) are believed to be modulated by additional regulatory proteins, which still have to be identified. Using biochemistry, molecularbiology and electrophysiology methods we demonstrate a protein-protein interaction between HCN2 and the K+ channel regulator protein 1, named KCR1. In coimmunoprecipitation experiments we show that KCR1 and HCN2 proteins are able to associate. Heterologously expressed HCN2 whole-cell current density was significantly decreased by KCR1. KCR1 profoundly suppressed IHCN2 single-channel activity, indicating a functional interaction between KCR1 and the HCN2 channel subunit. Endogenous KCR1 expression could be detected in adult and neonatal rat ventriculocytes. Adenoviral-mediated overexpression of KCR1 in rat cardiomyocytes (i) reduced If whole-cell currents, (ii) suppressed most single-channel gating parameters, (iii) altered the activation kinetics, (iv) suppressed spontaneous action potential activity, and (v) the beating rate. More importantly, siRNA- based knock-down of endogenous KCR1 increased the native If current size and single-channel activity and accelerated spontaneous beating rate, supporting an inhibitory action of endogenous KCR1 on native If. Our observations demonstrate for the first time that KCR1 modulates IHCN2/If channel gating and indicate that KCR1 serves as a regulator of cardiac automaticity. Citation: Michels G, Er F, Khan IF, Endres-Becker J, Brandt MC, et al (2008) K+ Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current If. PLoS ONE 3(1): e1511. doi:10.1371/journal.pone.0001511 INTRODUCTION Hyperpolarization-activated cation channels are found in a variety of cardiac cells and neurons [1–3]. These channels activate in response to hyperpolarization to generate an inward current termed If (‘‘funny’’) in cardiac cells, Ih (‘‘hyperpolarization’’- activated) in neurons, or Iq (‘‘queer’’). If has been proposed to contribute to pacemaker depolarization which generates rhythmic activity in spontaneously active cardiac cells [4,5] and neurons [6,7]. A family of four homologous hyperpolarization-activated, cyclic nucleotide-gated ion channel subunits (HCN1-4) have been identified [8–11]. In heterologous expression all HCN channels give rise to a hyperpolarization-activated inward current with similar but not identical characteristics compared to native If [8,9,12]. These observations suggest that HCN channel function is likely to be modulated by regulatory proteins and b-subunits in myocardial tissue. The K+ channel regulator 1 (KCR1), originally cloned from rat cerebellum, is a plasma membrane-associated protein with 12 putative transmembrane regions which is also expressed in rat cerebrum, and in rat and human heart [13,14]. The KCR1 protein can associate with rat ether-a`-go-go (EAG) and human ether-a`-go-go related (HERG) channel subunits [13–15]. Given the structural similarity and sequence analogy of HERG and HCN genes, we speculated that KCR1 might also interact with HCN channel subunits [8,9,12]. Therefore, we evaluated whether KCR1 and HCN2 proteins can associate. Secondly, we aimed to determine any possible functional modulation of IHCN2 and native If current characteristics by KCR1 in electrophysiological studies. Our results show that KCR1 and HCN2 proteins interact and demonstrate that KCR1 profoundly alters IHCN2 and If gating properties. Furthermore, KCR1 suppressed spontaneous rhythmicity in cardiocytes. Thus, our observations indicate that KCR1 serves as a regulatory protein of native If. RESULTS KCR1 and HCN2 associate in a protein complex To determine whether HCN and KCR1 gene products can form a protein complex, we prepared protein extracts from CHO cells cotransfected with KCR1 cDNA incorporating triple FLAG tags at the 59 end (pCFLAG3-KCR1) and HCN2 cDNA. Control cells were transfected with pCFLAG3-KCR1 alone or cotransfected with HCN2 and the empty FLAG-epitope containing vector. Input lysates were assayed in Western blots using an anti-HCN2 antibody to show successful production and detection of the HCN2 protein (Figure 1A). In addition, cell lysates were immunoprecipitated with anti-FLAG-Sepharose and then blotted using the anti-HCN2 antibody. Indeed, a band with the expected molecular mass of HCN2 was detected by the anti-HCN2 antibody in cells cotransfected with HCN2 and pCFLAG3- Academic Editor: Huibert Mansvelder, Vrije Universiteit Amsterdam, Netherlands Received July 30, 2007; Accepted December 22, 2007; Published January 30, 2008 Copyright: 2008 Michels et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant from the Deutsche Forschungs- gemeinschaft (Ho 2146/3-1), by Ko¨ ln Fortune, and by the Marga und Walter Boll- Stiftung. Funders were not involved in the design and conduct of the study, in the collection, analysis, and interpretation of the data, and in the preparation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: uta.hoppe@uni-koeln. de PLoS ONE | www.plosone.org 1 January 2008 | Issue 1 | e1511