. ... Table 1. Gating of single recombinant HCN2 and KCR1- . . cotransfected I .. . . . . . . . . . . . . . . . . . H.C..N.2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Parameter HCN2 control HCN2+KCR1 (1:1) HCN2+KCR1 (1:2) . . ... Open probability (%) 32.668.79 23.4613.14 0.9960.32* .... Availability (%) 78.566.10 54.6620.48 22.964.01* ... Mean open time (ms) 1.1560.15 0.7660.16 0.4460.06* . ... Mean closed time (ms) 2.9460.45 1.2360.44* 2.7860.61 .... Mean first latency (ms) 37.266.27 38.0617.14 42.965.94 ... Amplitude (pA) 22.1660.15 20.8660.06* 20.6860.01* . ... Conductance (pS) 34.662.431) 12.361.44* 8.3960.63* . ... Ipeak (fA) 8096170 3986204 43614* .... number of experiments 10 (6) 4 (4) 9 (4) . . .. Single-channel parameters of HCN2 (control) and KCR1-cotransfected IHCN2 . .. channels in CHO-cells. Holding potential 235 mV, test potential 290 mV. Ipeak .. was measured from ensemble average currents. For closed time and latency .. . analysis, only experiments containing just one detected open level were used .. for calculation. Numbers of experiments given in parentheses indicate number ... of experiments with only one channel in the patch. Pooled data are presented .. as mean6SEM. .. *p,0.05 vs. control. .. . 1)in this case n = 12 experiments were taken for conductance calculation. .. . doi:10.1371/journal.pone.0001511.t001 [5.4%], 34.68 [2.1%] (n = 6) vs. If+KCR1-tclosed values [ms]: 0.13 [48.3%], 0.92 [24.1%], 1.24 [17.9%], 5.76 [5.8%], 34.84 [3.9%] (n = 5), Figure 6C and 6D). These results suggest that endogenous KCR1 already modulated open kinetics of native If. KCR1 Modulates If Current Suppression of endogenous KCR1 increases If current density, single-channel activity of native If and spontaneous beating rate While alteration of If by KCR1 overexpression already indicated a direct interaction of HCN channels and KCR1 in native tissue, we further confirmed this finding by suppression of endogenous KCR1 using siRNA. Quantitative real-time PCR verified specific KCR1 knock-down by our siRNA in transfected CHO cells (normalized KCR1 mRNA: KCR1 10060.09%; KCR1+pENTR/U6- KCR1siRNA 34.860.03%; KCR1+pENTR/U6-LacZsiRNA 91.160.08%) and neonatal cardiomyocytes (normalized KCR1 mRNA: control 10060.07%; KCR1+pENTR/U6-KCR1siRNA 31.660.09%; KCR1+pENTR/U6-LacZsiRNA 96.560.1%). To evaluate the functional modulation of KCR1 knock-down we choose neonatal myocytes, because (i) these cells enable both analysis of KCR1-mediated effects on If current and spontaneous beating activity, and (ii) adult cardiomyocytes exhibit a high rate of dedifferentiation during culture for .48 h [22]. Compared to control conditions suppression of endogenous KCR1 significantly increased whole-cell If current density (6.0960.6 pA/pF n = 11; p = 0.04 vs. control, Figure 5A, 5C and 5D). Moreover, KCR1 knock-down markedly increased the single- channel activity and altered the gating behavior of native If (Figure 7, Table 2). In contrast to KCR1 overexpression, KCR1 knock-down shifted the voltage of the half-maximal activation to more positive values (244.560.9 mV, n = 15; p,0.05 vs. control; slope factor k 28.6163.8 mV; p = n.s vs. control, Figure 7B). Consistent with our observations in experiments with enhanced and heterologeous KCR1 expression, analyses of the multi-state gating revealed the Figure 5. KCR1 reduces current size of native If in neonatal rat cardiomyocytes. (A–C) Original whole-cell recordings of If in neonatal rat cardiocytes: (A) control, (B) KCR1-infected and (C) KCR1siRNA-infected. (D) Mean current densities of If in neonatal cells show that KCR1 overexpression reduced If, while suppression of endogenous KCR1 by KCR1siRNA significantly increased native If (*, p,0.001). For data see text. doi:10.1371/journal.pone.0001511.g005 PLoS ONE | www.plosone.org 6 January 2008 | Issue 1 | e1511