K+ Channel Regulator KCR1 Suppresses Heart Rhythm by Modulating the Pacemaker Current If

Abstract

Hyperpolarization-activated, cyclic nucleotide sensitive (HCN) channels underlie the pacemaker current If, which plays an essential role in spontaneous cardiac activity. HCN channel subunits (HCN1-4) are believed to be modulated by additional regulatory proteins, which still have to be identified. Using biochemistry, molecularbiology and electrophysiology methods we demonstrate a protein-protein interaction between HCN2 and the K+ channel regulator protein 1, named KCR1. In coimmunoprecipitation experiments we show that KCR1 and HCN2 proteins are able to associate. Heterologously expressed HCN2 whole-cell current density was significantly decreased by KCR1. KCR1 profoundly suppressed IHCN2 single-channel activity, indicating a functional interaction between KCR1 and the HCN2 channel subunit. Endogenous KCR1 expression could be detected in adult and neonatal rat ventriculocytes. Adenoviral-mediated overexpression of KCR1 in rat cardiomyocytes (i) reduced If whole-cell currents, (ii) suppressed most single-channel gating parameters, (iii) altered the activation kinetics, (iv) suppressed spontaneous action potential activity, and (v) the beating rate. More importantly, siRNA-based knock-down of endogenous KCR1 increased the native If current size and single-channel activity and accelerated spontaneous beating rate, supporting an inhibitory action of endogenous KCR1 on native If. Our observations demonstrate for the first time that KCR1 modulates IHCN2/If channel gating and indicate that KCR1 serves as a regulator of cardiac automaticity.

Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K + channels

“…Proteins were prepared using standard methods, and protein concentration was assayed with a commercial protein assay (BCA method; Pierce), as described previously (9,40). After standard Laemmli SDS/PAGE (12% wt/vol) and Western blotting (Tankblot system, nitrocellulose membrane; BioRad), proteins were detected using the following primary antibodies: Cx43 (1:500, rabbit polyclonal anti-rat total Cx43; Zytomed), Cx43 phosphorylated at serine 368 (p-Cx43Ser368, 1:500, rabbit polyclonal IgG; Cell Signaling), GSK3β (1:500, rabbit monoclonal antihuman; Cell Signaling Technology), GSK3β phosphorylated at serine 9 (1:500, rabbit monoclonal IgG; Cell Signaling Technology), GAPDH (1:200, rabbit polyclonal IgG anti-human; Santa Cruz Biotechnology, Inc.), Na + /K + -ATPase (1:1,000, rabbit polyclonal IgG anti-human α1 subunit; Cell Signaling Technology), cytochrome C oxidase IV (CCX IV, 1:500, rabbit polyclonal IgG; Abcam), manganese superoxide dismutase (MN-SOD, 1:1,000, rabbit polyclonal IgG; Upstate), and HRP-coupled anti-rabbit secondary antibody (1:2,000; Sigma-Aldrich).…”

Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes

“…Single-channel analysis was done using custom software only from 1-channel patches, as previously reported (13,37,43,44). Linear leak and capacity currents were digitally subtracted using the average currents of nonactive sweeps.…”

“…This notion was further supported by an additive effect of 43 GAP27 with the genetic Cx43 knockdown, i.e., the Cx43 mimetic peptide abolished diazoxide-mediated K ATP channel activation in Cx43 +/-mice ( Figure 3C and Table 2). Inhibition of mitoK ATP channels by MgATP (Cx43 +/-Po,total, 0.27% ± 0.09%, n = 4) and glibenclamide was retained in the presence of carbenoxolone or 43 GAP27 in Cx43 +/-mice and in Cx43 Cre-ER(T)/fl + 4-OHT mice ( Figure 3D and Tables 1-3).…”

“…(A) The diazoxide effect on mitoKATP channel activity (at -60 mV) was reduced in wild-type mice by carbenoxolone (10 μM) (upper left) and 43 conducting pore of mitoK ATP channels or might transfer cytoprotective signaling to these channels. To define the role of mitochondrial Cx43, we used 4 complementary approaches, i.e., single-channel recordings of: wild-type mitoplasts in the presence of (a) the connexin inhibitor carbenoxolone (10 μM) (19) (Figure 3, A-E, and Tables 1-3), indicating that the effect of diazoxide on mitoK ATP channels is mediated via Cx43.…”

A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins

“…HCN channel properties have been shown to be altered by interaction 64 with caveolin-3 (cav-3), MiRP1, KCR1 and SAP97 proteins [10][11][12][13][14][15].…”