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.
Highlights
Hyperpolarization-activated cation channels are found in a variety of cardiac cells and neurons [1,2,3]
Our results show that K+ channel regulator 1 (KCR1) and HCN2 proteins interact and demonstrate that KCR1 profoundly alters IHCN2 and If gating properties
To determine whether HCN and KCR1 gene products can form a protein complex, we prepared protein extracts from CHO cells cotransfected with KCR1 cDNA incorporating triple FLAG tags at the 59 end and HCN2 cDNA
Summary
Hyperpolarization-activated cation channels are found in a variety of cardiac cells and neurons [1,2,3]. If has been proposed to contribute to pacemaker depolarization which generates rhythmic activity in spontaneously active cardiac cells [4,5] and neurons [6,7]. In heterologous expression all HCN channels give rise to a hyperpolarization-activated inward current with similar but not identical characteristics compared to native If [8,9,12]. These observations suggest that HCN channel function is likely to be modulated by regulatory proteins and b-subunits in myocardial tissue
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