KCR1 Modulates If Current . ... Table 2. Single-channel parameters of native If in adult and neonatal cardiomyocytes modulated by KCR1 and KCR1siRNA .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Adult myocyte If Adult myocyte If Neonatal myocyte If Neonatal myocyte If Neonatal myocyte If ... Parameter control KCR1-infected control KCR1-infected KCR1siRNA-infected . ... Open probability (%) 4.1060.74 0.9460.24* 9.1261.72# 4.0561.09* 16.363.24*# . ... Availability (%) 45.264.08 21.862.97* 41.268.46# 20.465.11* 50.267.55# . ... Mean open time (ms) 0.5560.06 0.5660.05 0.4360.06 0.4060.05 0.4760.05 .... Mean closed time (ms) 1.8560.28 2.2260.10 2.4160.34 3.9460.65 2.1360.37# ... Mean first latency (ms) 53.966.80 62.764.35 40.565.51 46.367.49 28.664.22# . ... Amplitude (pA) 20.8560.05 20.5960.02* 20.8660.02# 20.5960.02* 21.2360.11*# .... Conductance (pS) 8.8160.25 6.1060.26* 7.0160.49# 4.2760.31* 13.660.94*# ... Ipeak (fA) 7069 2762* 49613 2367 102627 . ... number of experiments 10 (6) 9 (5) 12 (5) 10 (6) 9 (5) . . . . Modulation of single-channel parameters of native If (control) by KCR1 overexpression (KCR1-infected) and knock-down of endogenous KCR1 (KCR1siRNA-infected) in . .. adult and neonatal cardiomyocytes. 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; .. #p,0.05 vs. KCR1-infected. .. . doi:10.1371/journal.pone.0001511.t002 additional b-subunits and regulatory proteins in native tissue. Recent studies demonstrated that HCN channels associate with several modulating proteins [26–31]. KCNE2 enhances the expression and accelerates activation of IHCN [26–28]. Filamin A modifies HCN1, but not HCN2 or HCN4 channel activity and distribution on the cell membrane [29]. Moreover, TPR- containing Rab8b interacting protein [30], tamalin, S-SCAM and Mint2 scaffold proteins [31] were reported to affect HCN trafficking and, thus, IHCN current density. However, since these effects are not sufficient to explain If properties in native tissue, it is likely that HCN currents are being modulated by one or more additional proteins. Using immunoprecipitation studies we now show that KCR1 and HCN2 proteins associate. It is well known that regulatory proteins can affect both expression and gating of pore forming a-subunits. Suppression of recombinant HCN2 single-channel activity by KCR1 clearly demonstrated a functional effect of KCR1 on HCN channel behavior. More importantly, knock-down of endogenous KCR1 also profoundly modulated native If function in ventriculo- cytes. Loss of KCR1 lead to (i) an increase of If current density, (ii) a higher activation of If on single-channel level, and (iii) a significant elevation of the spontaneous beating rate. Currently, two distinct ranges of single channel conductances for native If and cloned HCN channels have been reported depending on the recording technique (low conductance currents of 1–1.5 pS using a corner frequency of 80–800 Hz [17,18,20] vs. an approximately 10-fold higher conductance using a filtering of 2000 Hz [16,19]), as has been discussed previously [32]. In the present study we further confirmed that current openings in our recordings indeed were produced by HCN channels as we demonstrated (i) a typical increase of channel activity by the application of cAMP to the inner side of inside-out patches, and (ii) a characteristic channel inhibition by the If/HCN specific blocker ivabradine. Moreover, we could clearly show time- and voltage- dependence of HCN single-channel recordings obtained from multi- channel patches which were comparable to macroscopic If/IHCN currents. Given the evidence for cooperative gating between single HCN channels [20], expectedly, the sum of recordings from one- channel patches in the present study and in our previous report [16] did not result in average currents with identical kinetics compared to macroscopic If/IHCN, emphasising that for the evaluation of If/IHCN averages it is essential to distinguish between recordings obtained from patches with one individual channel vs. multi-channel patches. While differences of If/IHCN conductance in part might be caused by distinct recording techniques or by the expression of different HCN channel isoforms, it might also indicate HCN modulation by endogenous KCR1 within native channel complexes. Notably, in the present study single-channel parameters of native If more closely resembled those of HCN2+KCR1 than HCN2 alone (Table 1 and 2), like for instance single-channel conductance in ventriculocytes (7– 9 pS). A similar single-channel conductance (7–10 pS) has been reported for recordings of If in neurons [19,33], which also express KCR1 [13], supporting the hypothesis of potential If modulation by KCR1 in native tissue. Beside single-channel conductance a possible interaction of endogenous KCR1 and HCN was especially evident for single-channel open kinetics. While four open states could be obtained for HCN2 alone in heterologous expression, KCR1 reduced HCN2 open kinetics to only three states, which was however similar to native If. Consistent with this notion, knock-down of endogenous KCR1 increased kinetics of native If to four open states and increased single-channel conductance. These observations further indicate that KCR1 functionally interacts with native If in cardiac tissue. Interestingly, KCR1 overexpression shifted the voltage-depen- dence of If activation to more negative potentials, whereas KCR1 knock-down resulted in a shift of the half-maximal activation to more positive values. Low expression levels of KCR1 in sinus node cells might contribute to larger If current size and more positive voltages of mid-activation of If, whereas higher expression of KCR1 in the atrioventricular node and working myocardium could explain much smaller If currents and the more negative threshold potentials of If activation in these regions. However, it should always be acknowledged that (i) the regional distribution of HCN channel isoforms (e.g. HCN4 as the predominant isoform in sinoatrial node region), (ii) the variety of heteromultimerization, and (iii) the influence of other known/unknown modulating proteins together determine the biophysical behavior of native If. This might explain why single channel conductance of native If in sinoatrial node cells was not reported to be higher than in working myocardium or neurons [16,17,19,33]. KCR1 inhibited single-channel activity and seemed to keep HCN channels in a deeper closed-state indicating a functional PLoS ONE | www.plosone.org 8 January 2008 | Issue 1 | e1511