KCR1 Modulates If Current Figure 6. Effect of KCR1 on single native If channel gating in one-channel patches. (A) Comparison between single native If channels of control adult ventriculocytes (left) and KCR1-infected cells (right). Recording technique as in Figure 3. Scale bars, 50 ms, 2.5 pA for unitary current traces, and 1 s, 60 fA for ensemble average current. (B) KCR1 significantly shifted If 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 exhibited no effect on the number of native If open states. Number of open events (square root) were plotted against the logarithmically binned open time durations for If alone and with exogenous KCR1 (pooled one- and multi [n#3]-channel experiments). (D) Closed- time histograms: KCR1 did not affect the number of native If closed states. Number of closed events (square root) were plotted against the logarithmically binned closed time durations for If alone and with exogenous KCR1 (pooled one-channel experiments only). doi:10.1371/journal.pone.0001511.g006 opposite effect by KCR1 knock-down on native If, i.e. an increase in the number of open states and a loss of one closed-state (topen values of If-KCR1siRNA [ms]: 0.26 [46.4%], 1.19 [10.0%], 1.26 [25.7%], 3.21 [17.9%] (n = 9); tclosed values of If-KCR1siRNA [ms]: 0.10 [79.9%], 8.38 [20.1%] (n = 5); Figure 7C and 7D). To evaluate a possible functional modulation of KCR1 in spontaneously active tissue action potential recordings were performed in spontaneously beating neonatal cardiocytes. Control cultures beat spontaneously with a mean rate of 80.465.8 bpm (n = 17). Cycle length tended to vary from beat to beat (Figure 8A). Maximal diastolic potential was 256.463.5 mV. KCR1 overex- pression entirely suppressed beating activity in neonatal cardio- cytes (n = 20; maximal diastolic potential 258.662.7 mV; p = n.s. vs. control). Figure 8B illustrates a representative recording of an action potential that was induced artificially by a short depolarizing current pulse in a KCR1-infected myocyte. Con- versely, knock-down of endogenous KCR1 by siRNA resulted in an acceleration of the beating rate to 100.264.6 bpm (n = 17; p,0.005 vs. control; maximal diastolic potential 255.863.3 mV; p = n.s. vs. controls, Figure 8C and 8D). This obvious inhibitory effect of KCR1 on action potential frequency further supports the critical contribution of If to spontaneous beating activity of neonatal cardiocytes [4] and the modulating effect of KCR1 on native If and automaticity. DISCUSSION In addition to the pore forming a-subunit Na+, Ca2+ and K+ channels incorporate modulatory b-subunits, as well as scaffolding proteins, chaperones, cytoskeletal elements, and Ca2+-sensing proteins in their higher order structures [23–25]. KCR1 is a membrane-associated protein with 12 putative transmembrane regions [13,14]. In the present study KCR1 expression was demonstrated in neonatal and adult ventriculocytes. By coimmu- noprecipitation KCR1 was shown to form protein complexes with HCN2. KCR1 markedly altered IHCN and If whole-cell currents, single-channel activity and gating, and suppressed spontaneous action potential activity, indicating a functional interaction with HCN channels. Heterologously expressed HCN channels result in hyperpolar- ization-activated inward currents with similar, however, not identical properties compared to native If [8,9,12]. These observations suggest that HCN subunits are modulated by PLoS ONE | www.plosone.org 7 January 2008 | Issue 1 | e1511