KCR1 Modulates If Current Figure 4. Effect of KCR1 on single recombinant HCN2 channel gating in one-channel patches. (A) Comparison of single recombinant HCN2 channels transfected in CHO cells alone (left) and with KCR1 (right). Middle, 20 consecutive single traces of each channel without and with KCR1. Single channels were hyperpolarized at continuous pulse mode for a total duration of 3 s (206150 ms sweeps), with a holding potential of 235 mV and a test potential of 290 mV. Bottom, ensemble average current of one consecutive sweep of 3 s pulse duration. Scale bars, 50 ms, 6 pA (unitary current traces) for HCN2 alone (left) and 2 pA when co-transfected with KCR1 (right), or 1 s, 0.5 pA (ensemble average current) for HCN2 and HCN2+KCR1. (B) KCR1 significantly shifted IHCN2 activation to more negative potentials. Channel activation was measured by the parameter availability, plotted against the test potential and then determined by using the Boltzmann function. For data see text. (C) Open-time histograms: KCR1 reduced the number of HCN2 open states. Number of open events (square root) were plotted against the logarithmically binned open time durations for HCN2 alone and HCN2+KCR1 (pooled one- and multi [n#3]-channel experiments). (D) Closed-time histograms: KCR1 did not affect the number of HCN2 closed states. Number of closed events (square root) were plotted against the logarithmically binned closed time durations for HCN2 alone and HCN2+KCR1 (pooled one-channel experiments only). doi:10.1371/journal.pone.0001511.g004 adenoviral vectors expressing EGFP alone to 0.960.1 pA/pF (n = 9) in KCR1-infected cells when measured at 2130 mV (p,0.001) (Figure 5A, 5B and 5D). Similarly, If current size in adult rat ventricular myocytes was significantly suppressed from 3.360.5 pA/pF (n = 11) in controls to 0.3860.08 pA/pF (at 2130 mV; n = 10) in KCR1-infected myocytes (p,0.001). Direct modulation of If by KCR1 overexpression was further analysed in single-channel recordings. Notably, single-channel properties of native If in control cells more closely resembled those of heterologously expressed HCN2+KCR1 than of IHCN2 alone (Figure 6A, Table 2), indicating a possible modulation of HCN subunits by endogenous KCR1 in native cells. KCR1 further suppressed single-channel availability, open probability, and single-channel amplitude and conductance of native If (Figure 6A and 7A; Table 2), and shifted the voltage of half-maximal activation to more negative values (adult ventriculocytes: from 266.364.5 mV, n = 5, to 295.863.8 mV, n = 7; p,0.001, Figure 6B; neonatal ventriculocytes: from 256.461.3 mV, n = 12, to 281.363.2 mV, n = 10; p,0.0001, Figure 7B), without significantly affecting the slope factor k for adult (control: 211.962.1 mV, KCR1: 217.863.1 mV), and neonatal ventri- culocytes (control: 212.163.9 mV, KCR1: 216.764.8 mV). These data indicate that KCR1 modulates single-channel behavior of If in cardiac tissue. While KCR1 reduced the number of HCN2 open states from four to three in the heterologous expression system (CHO cells), in which no endogenous KCR1 was detectable, KCR1 had no effect on gating kinetics of native If in cardiomyocytes. In neonatal ventriculocytes we detected only three open and three closed states (If-topen values [ms]: 0.26 [58.9%], 0.87 [32.8%], 4.54 [8.3%] (n = 12) vs. If+KCR1-topen values [ms]: 0.25 [47.8%], 1.22 [43.1%], 5.56 [9.1%] (n = 10); If-tclosed values [ms]: 0.18 [57.5%], 1.57 [33.6%], 12.7 [8.9%] (n = 5) vs. If+KCR1-tclosed values [ms]: 0.15 [45.1%], 1.13 [37.3%], 16.5 [17.6%] (n = 6), Figure 7C and 7D), whereas in adult ventriculocytes we observed two more closed state (If-topen values [ms]: 0.23, 0.94, 4.64 (n = 7) vs. If+KCR1-topen values [ms]: 0.18, 0.99, 4.59 (n = 6); If-tclosed values [ms]: 0.16 [60.2%], 0.99 14.7%], 1.21 [17.6%], 5.47 PLoS ONE | www.plosone.org 5 January 2008 | Issue 1 | e1511