KCR1 Modulates If Current Figure 3. Electrophysiological and pharmacological properties of IHCN/If on single-channel level from inside-out recordings. (A) Representative HCN2 single-channel currents (sampling frequency: 5 kHz, corner frequency: 1 kHz) of inside-out recordings from one- and multi-channel (n,50 channels) patches at different test-potentials (290 to 2130 mV). (B) Pharmacological characteristics of HCN2 single-channels (test potential: 290 mV, holding potential: 235 mV). Ivabradine (50 mM) blocks HCN2 single-channel current during repetitive activation/deactivation steps (290 mV, 150 ms/+10 mV, 600 ms). The observations that ivabradine significantly reduced the open probability (25.867.07% vs.6.0863.45%, n = 4, p,0.05), the mean open time (1.0360.12 ms vs. 0.6160.16 ms, n = 4, p,0.05) and the availability (75.9610.1% vs. 25.366.98%, n = 4, p,0.05) suggests an open-channel blockade by a fast and a slow gating mechanism. cAMP induced an increase of the channel activity (for data, see text). The data were sampled at 10 kHz and filtered at 2 kHz. (C) Effect of ivabradine (Iva) and cAMP on single-channel activity. Open probability (Popen) decreased after ivabradine (50 mM) and increased after cAMP (1 mM) application, respectively. Data recorded as in Figure 3B. doi:10.1371/journal.pone.0001511.g003 IHCN2+KCR1(1:2) was 258.564.8 mV, n = 6, vs. 289.764.9 mV, n = 5, p = 0.001, with the slope factor k being unchanged: 215.863.0 mV and 212.462.7 mV, respectively; Figure 4B). We recently revealed an allosteric multi-state gating model comprising at least four open and five closed states of single HCN2 channel recordings [16,21]. In the present study KCR1 altered HCN2 channel open kinetics to only three open states (HCN2- topen values [ms]: 0.13 [47.5%], 0.86 [33.5%], 2.24 [18.5%], 5.93 [0.5%] (n = 8) vs. HCN2+KCR1 (1:1)-topen values [ms]: 0.13 [54.7%], 0.87 [38.5%], 2.72 [6.8%] (n = 4) and HCN2+KCR1 (1:2)-topen values [ms]: 0.13 [77%], 0.69 [21.9%], 2.36 [1.1%] (n = 5)), while not significantly affecting the five closed states (HCN2-tclosed values [ms]: 0.15 [55.7%], 0.90 [28.4%], 3.36 [5.5%], 5.52 [4.1%], 21.0 [6.3%] (n = 5) vs. HCN2+KCR1 (1:1)- tclosed values [ms]: 0.12 [64.9%], 0.55 [18.9%], 0.97 [12%], 2.96 [3.8%], 27.0 [0.4%] (n = 4) and HCN2+KCR1 (1:2)-tclosed values [ms]: 0.14 [76.3%], 0.83 [16.8%], 1.78 [3.1%], 7.80 [1.3%], 37.92 [2.5%] (n = 4) (Figure 4C and 4D). KCR1 is highly expressed in the atrioventricular- node While functional interaction of HCN and KCR1 gene products in heterologous mammalian systems is interesting, it is even more important whether KCR1 is present in cardiac tissue and might also modulate native If. To clarify this situation we chose neonatal and adult rat ventriculocytes. To omit any interference of non-cardiac cells, we performed reverse transcriptase PCR analysis of single neonatal and adult rat ventricular myocytes. Indeed, in both cell types the expected band could be detected (Figure 1B), demonstrat- ing endogenous expression of KCR1 in native cardiac cells. To analyze regional expression of KCR1, we determined KCR1 mRNA levels of freshly dissected samples from different parts of adult rat and pig hearts by quantitative real-time PCR. In both species we consistently obtained highest KCR1 expression in the atrioventricular node (rat: 536648%, n = 3; pig: 637652%, n = 3; p,0.05 vs. all other regions) and lowest levels of KCR1 in the sinus node (rat: 46.368.10%, n = 3; pig: 52.764.98%, n = 3; p,0.05 vs. all other regions) (Figure 1C). KCR1 overexpression suppresses current density and single-channel activity of native If Considering our results in heterologous expression, overexpression of a construct carrying KCR1 should reduce If current density in myocardium if HCN and KCR1 also interact in native tissue. In neonatal ventriculocytes If density was indeed markedly reduced from 4.760.3 pA/pF (n = 14) in control cells infected with PLoS ONE | www.plosone.org 4 January 2008 | Issue 1 | e1511