Abstract-The HCN family of ion channel subunits underlies the currents I f in heart and I h and I q in the nervous system.In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a ␤ subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a ␤ subunit for the cardiac delayed rectifier I Kr , these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001;88:e84-e87.)Key Words: HCN family Ⅲ MiRP1 Ⅲ KCNE family Ⅲ ␤ subunit T he HCN (hyperpolarization-activated cyclic nucleotidegated) family of ion channel subunits has been identified as the molecular correlate of the currents I f in heart and I h and I q in neurons. [1][2][3] However, several ion channels are heteromultimers of a large ␣ subunit (like the HCN family members) and smaller ␤ subunits. The cardiac delayed rectifiers I Kr 4 and I Ks 5 are examples of this basic principle. Their ␣ subunits derive from the ERG and KCNQ families, respectively, but both also contain ␤ subunits from the KCNE family of single transmembranespanning proteins called minK and minK-related peptides (MiRPs). In this study, we report that MiRP1 enhances the expression and speeds the kinetics of activation of the HCN family of channel subunits. From immunoprecipitation experiments, we show that it most probably forms a complex with HCN1. Using RNase protection assays (RPAs), we demonstrate that MiRP1 mRNA is prevalent in the primary cardiac pacemaking region, the sinoatrial (SA) node, and barely detectable in ventricle. Cardiac pacemaker activity is generated by a narrow balance of inward (I f ) and outward (I Kr ) currents. Our results demonstrate for the first time the potential importance of a single ␤ subunit in simultaneously regulating both the expression and gating of both inward and outward cardiac pacemaker channels. Materials and Methods Heterologous Expression in Xenopus OocytescRNA encoding mouse HCN1 or HCN2, rat MiRP1 with or without an HA tag at the carboxy-terminal, and rat minK were transcribed using the mMessage mMachine kit (Ambion). Xenopus laevis oocytes were isolated, injected with 2 to 5 ng (50 to 100 nL) of cRNA, and maintained in Barth medium at 18°C for 1 to 3 days. For experiments using both HCN1 or HCN2 and MiRP1 or minK, the respective cRNAs were injected in a 1:0.04 to 1 ratio. Electrophysiological studies on oocytes used the 2-microelectrode voltage clamp. The extracellular recording solution (OR2) contained, in mmol/L, NaCl 80, KCl 2, MgCl 2 1, and Na-HEPES 5 (pH 7.6). Group data are presented as meanϮSEM. Tests of statistical significance for midpoint and slope of activation curves were performed using unpaired Student's t tests. PϽ0.05 is considered significant. RNase Protection AssaysThe procedures for the preparation of total RNA from rabbit he...

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