HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET 2 ), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET 2 data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue. Hyperpolarization-activated cyclic nucleotide-modulated (HCN)2 channels, which underlie hyperpolarization-activated or funny currents (I h or I f ) in excitable cells, are thought to be made up of subunits that assemble as tetramers to form functional channels (1). Four mammalian HCN isoforms (HCN1 to -4) (2-6) possess various overlapping patterns of expression in the heart and throughout the central nervous system, suggesting that they form heteromeric channels in these tissues (1,7,8). Previous studies suggest that the following combinations of HCN isoforms co-assemble and form functional channels in heterologous expression systems: HCN1 with HCN2 (9 -12) and HCN1 with HCN4 (11). On the other hand, whether HCN2 and HCN4 isoforms co-assemble and form functional channels has not been shown and is an important objective of the present experiments.The best evidence for co-assembly of HCN2 and HCN4 in native tissue comes from studies in the embryonic heart and adult thalamus. In the embryonic mouse heart, mRNA for HCN2 and both mRNA and protein for HCN4 have been found (13-16). Knock-out of HCN4 reduces, but does not abolish, I f and speeds up rates of I f activation in cardiomyocytes, consistent with the presence of other HCN isoforms in these cells (16). Immunohistochemical approaches in rats and mice have demonstrated HCN2 and HCN4 protein in thalamocortical relay nuclei (17, 18) and colocalization in cells of the ventrobasal complex and reticular nucleus of the thalamus (19). Knock-out of HCN2 reduces, I f in thalamocortical neurons, consistent with the presence of other HCN isoforms in these cells (20).Co-assembly of HCN2 and HCN4 is supported by evidence of interaction of the two isoforms in heterologous expression...

Proteolytic Processing of HCN2 and Co-assembly with HCN4 in the Generation of Cardiac Pacemaker Channels

“…This conclusion is in accord with previous immunohistochemical findings showing that HCN2 and HCN4 are co-localized in (embryonic) mouse heart (22). The results here importantly extend these earlier findings with the demonstration that HCN2 and HCN4 can be co-immunoprecipitated from heart.…”

“…together with heteromeric HCN2: HCN4 channels, in the myocardium (22). Consistent with previously reported findings (15,22), results are also presented here showing that both HCN2 and HCN4 can form homomeric HCN channels in heterologous cells when expressed alone and that the time-and voltage-dependent properties of the (homomeric) currents are distinct (Fig.…”

“…Consistent with this suggestion, resonance energy transfer experiments suggest that homomeric and heteromeric HCN channels are present in native tissues (22).…”

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

“…The number of HCN channels on the cell surface is a critical determinant of beating frequency in cardiac conduction tissue. HCN isoforms and HCN-mediated currents (I h ) are upregulated in the embryonic and neonatal ventricle (24,45,53,56), in the neonatal sinoatrial node (1), and in beating embryonic stem cells during development in culture (27,36,40,41). However, the factors that determine HCN channel supply at the cell surface during cardiac development have been studied in only a limited fashion.…”

Biological Pacemaker – Main Ideas and Optimization

“…I f is encoded by both HCN2 and HCN4 in the ventricles (Shi et al, 1999;Stillitano et al, 2008). Recent evidence has shown that they can form heteromeric channels in vitro and in vivo producing an I f with physiological activation (Much et al, 2003;Whitaker et al, 2007;Zhang et al, 2009). The respective roles for I f and I K1 in the induced ventricular pacemaker activity have recently been evaluated in the guinea pig left ventricular myocytes overexpressing either HCN1-ΔΔΔ or Kir2.1 (Chan et al, 2009).…”