MiRP1 Modulates HCN2 Channel Expression and Gating in Cardiac Myocytes

Abstract

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotidegated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between ؊60 and ؊95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a ␤ subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.MinK-related protein (MiRP1 or KCNE2) is purported to be a ␤ subunit for several voltage-gated potassium channels, including the rapid delayed rectifier (1), slow delayed rectifier (2), and transient outward current (3). In addition, we have reported that it can act as a ␤ subunit for the hyperpolarizationactivated, cyclic nucleotide-gated (HCN) 1 family of pacemaker channels (4). We found that co-expression of MiRP1 with HCN1 or HCN2 in Xenopus oocytes resulted in larger and more rapidly activating currents than when either HCN isoform was expressed alone but that MiRP1 did not shift the midpoint of activation of either isoform. Further, co-immunoprecipitation experiments involving HA-tagged MiRP1 and HCN1 demonstrated that the two proteins interact when co-expressed in oocytes.A more recent study demonstrated MiRP1 interaction with the HCN4 isoform. Here, co-expression with MiRP1, either in oocytes or Chinese hamster ovary cells, also resulted in increased current amplitude, but this was associated with slowing of kinetics and a negative shift of the midpoint of activation (5). However, another laboratory did not detect any effect of MiRP1 when co-expressed in HEK293 cells with either HCN4 or an HCN1-HCN4 tandem construct (6). Finally, a study of MiRP1 and HCN2 co-expression in Chinese hamster ovary cells reported a reduced time-dependent current component and increased instantaneous ...

Hyperpolarization-Activated Currents Regulate Excitability in Stellate Cells of the Mammalian Ventral Cochlear Nucleus

“…In hippocampal pyramidal cells ZD7288, at 10 -20 M, blocked ϳ18% of the resting conductance (Gasparini and DiFrancesco 1997). Other authors, however, did not observe an instantaneous, ZD7288-sensitive conductance in thalamic relay neurons (Luthi et al 1998) and with coexpression of HCN with the MiRP1 protein in cardiac sinoatrial cells (Qu et al 2004).…”

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels

“…The β accessory subunit MiRP1 changes the activation kinetics of the current in opposite ways depending on the HCN isoform. Although it has been reported to increase the activation kinetics and the currents of HCN1 and 2 [43,44] , it decreases the activation kinetics but increases the I f current when it is associated with HCN4 [45] . Other HCN regulatory mechanism have been described, including phosphatidylinositol 4,5-bisphosphate (PIP 2 ) modulation [46] or the direct actions of kinases [47] and phosphatases [48,49] .…”

Regulation of HCN Channel Surface Expression by a Novel C-Terminal Protein-Protein Interaction

“…The heterogeneity of I h is attributable to the presence of four closely related HCN genes (HCN1-4) that generate I h channels with distinct biophysical properties and tissue distribution (Santoro et al, 1998(Santoro et al, , 2000Moosmang et al, 2001). Recently, the integral membrane protein MiRP1 (Mink-related protein 1) has been found to serve as a ␤ subunit for HCN channels (Yu et al, 2001;Qu et al, 2004).…”