KCR1, whereas it could not be coimmunoprecipitated from extracts containing pCFLAG3-KCR1 alone or HCN2 and the empty FLAG-epitope containing vector (Figure 1A). These results indicate that HCN2 and KCR1 associate in protein complexes in mammalian cells, while excluding any unspecific detection of KCR1 by the anti-HCN2 antibody and any unspecific coimmu- noprecipitation of the FLAG-epitope and HCN2. In addition, unspecific coimmunoprecipitation by the anti-FLAG-Sepharose could be excluded by mock immunoprecipitation with normal A-Sepharose (Figure 1A). KCR1 reduces HCN2 current size and profoundly modulates HCN2 channel gating To evaluate a possible functional interaction of HCN2 and KCR1, the effect of KCR1 on IHCN2 was analysed. Whole-cell IHCN2 currents were recorded from CHO cells transfected with HCN2 (0.25 mg/well) alone or together with KCR1 (ratio 1:1, 1:2 or 1:3; total cDNA amount adjusted to 1 mg/well with the unrelated channel subunit Kv1.3AYA in all experiments). RT-PCR revealed no detectable KCR1 in non-transfected CHO cells (Figure 1B). Representative current recordings (Figure 2A) and mean current densities (Figure 2B) show that IHCN2 (79.6613.9 pA/pF at 2130 mV, n = 9) was significantly decreased by KCR1 (15.86 10.4 pA/pF, ratio 1:1, n = 14; 3.662.1 pA/pF, ratio 1:2, n = 10; 2.560.9 pA/pF, ratio 1:3, n = 17; p,0.001). Moreover, KCR1 significantly shifted half-maximal activation of IHCN2 (2102.06 KCR1 Modulates If Current 2.1 mV) to more negative potentials (2109.862.0 mV for HCN2+KCR1, ratio 1:1; p = 0.048; n = 12). To further confirm a functional modulation of HCN2 channel properties by KCR1 additional single-channel recordings were performed. Available data about HCN single-channel properties vary depending on cell-type and recording technique [16–20]. While we previously already showed characteristic stimulation of single HCN2 channels by forskolin [16], we now further proved that the channel openings in our experiments were produced by HCN channels demonstrating (i) a time- and voltage-dependent kinetic similar to macroscopic IHCN/If current from HCN2 multi- channel recordings, which allows in contrast to ‘‘pure’’ one- channel recording cooperative HCN channel gating (Figure 3A), (ii) a HCN2 single-channel conductance of these multi-channel patches (24.064.62 pS, n = 3) comparable to our cell-attached + recordings (considering the symmetrical K -solution in inside-out recordings) (Figure 3A), (iii) a typical increase of the open probability upon cAMP application in inside-out recordings (control: 24.7611.1% vs. Br-cAMP 1 mM: 51.0614.1%, n = 4, p,0.05) (Figure 3B and 3C), and (iv) a reduction of HCN2 open probability by the specific If inhibitor ivabradine (50 mM) (25.867.07% vs. 6.0863.45%, n = 4, p,0.05) (Figure 3B and 3C). Single-channel characteristics of cells cotransfected with HCN2 and KCR1 were distinct from cells expressing HCN2 alone. KCR1 profoundly reduced single-channel activity of IHCN2 (Figure 4A; Table 1). Moreover, KCR1 caused a hyperpolarization shift of the voltage of half-maximal IHCN2 activation (V0.5 for IHCN2 and Figure 2. KCR1 suppresses recombinant IHCN2 current density. (A) Representative HCN2 whole-cell currents in absence and presence of KCR1. (B) Mean current densities of heterologously expressed HCN2 alone or together with KCR1 demonstrate that KCR1 significantly (*, p,0.001) reduced current density of recombinant IHCN2. For data see text. doi:10.1371/journal.pone.0001511.g002 PLoS ONE | www.plosone.org 3 January 2008 | Issue 1 | e1511