KCR1 Modulates If Current Figure 7. Effect of KCR1 and KCR1siRNA on single native If channel gating in one-channel patches. (A) Comparison between single native If channels of control neonatal ventriculocytes (left), KCR1-infected (middle) and KCR1siRNA-infected cells (right). Recording technique as in Figure 3. Scale bars, 50 ms, 3 pA for unitary current traces, and 1 s, 20 fA for ensemble average current. (B) Enhanced expression and knock-down of KCR1 significantly shifted the half-maximal activation of If to more negative and more positive potentials, respectively. 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 exhibited no effect on the number of native If open states, whereas KCR1siRNA induced an increase of the number of open states. Number of open events (square root) were plotted against the logarithmically binned open time durations for If alone and with exogenous KCR1 or KCR1siRNA (pooled one- and multi [n#3]-channel experiments). (D) Closed-time histograms: KCR1 did not affect the number of native If closed states, while KCR1 knock-down resulted in a loss of one closed state. Number of closed events (square root) were plotted against the logarithmically binned closed time durations for If alone and with exogenous KCR1 or KCR1siRNA (pooled one-channel experiments only). doi:10.1371/journal.pone.0001511.g007 alteration of HCN channel subunits as underlying mechanism of KCR1-mediated modulation of If and cardiac automaticity. Further potential KCR1-induced regulatory mechanisms might involve alteration of HCN trafficking and cell-surface stability. Although such effects could not explain the changes of If single- channel gating, they might contribute to the observed KCR1- mediated actions and will be addressed in future studies. KCR1 significantly suppressed spontaneous beating activity of neonatal cardiocytes, implying that KCR1 contributes to modu- lation of the spontaneous activity in cardiac cells. Interestingly, both KCNE2 and KCR1 originally were identified as potential regulatory subunits of HERG, the pore forming a-subunit of IKr [14,34]. These and our observations suggest, that both proteins may have a prominent role for the balance of inward (If) and outward (IKr) currents involved in cardiac pacemaker activity. Given these obvious inhibitory effects of KCR1 on HCN channels and native If, alterations of KCR1 expression might cause changes in heart rate or ectopic spontaneous depolarizations in patients, though our in-vitro observations cannot readily be generalized to humans in-vivo. Interacting proteins like KCR1 inhibiting If and, thus, depressing spontaneous activity may provide a novel therapeutic target of cardiac arrhythmias. MATERIALS AND METHODS Plasmids, Small Interfering RNA and Adenovirus Preparation The expression plasmids pAdCGI-HCN2 encoding the full-length sequence of mHCN2, pCGI-Kv1.3AYA and pAdCGI have been described [4,35]. The full-length coding sequence of rat KCR1 (kindly provided by Dr. H. Higashida, Kanazawa University, Japan) was cloned into the multiple cloning site of pAdCGI, to give pAdCGI-KCR1. The red fluorescent protein coding sequence (Red) of psDRed-Express-1 (Clontech, Palo Alto, CA) was cloned PLoS ONE | www.plosone.org 9 January 2008 | Issue 1 | e1511