Properties of Hyperpolarization-Activated Pacemaker Current Defined by Coassembly of Hcn1 and Hcn2 Subunits and Basal Modulation by Cyclic Nucleotide

Abstract

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5–10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10–20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose–response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of Ih in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.

Formation of heteromeric hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the hippocampus is regulated by developmental seizures

“…While the ability of HCN1 and HCN2 to co-assemble into functional channels has been demonstrated (Chen et al, 2001b;Er et al, 2003;Much et al, 2003;Proenza et al, 2002;Ulens and Tytgat, 2001;Xue et al, 2002), we find only modest co-association of HCN1/HCN2 in the 'normal' hippocampal formation of both developing and adult rats (control groups, Figs. 1 and 5), consistent with the single report on whole mouse brain (Much et al, 2003).…”

“…Whereas in vivo evidence for this abundance-dependent dynamic interaction of HCN channels isoforms is not yet available, in vitro studies, transfecting different ratios of HCN1/HCN2 constructs into heterologous expression systems, support this notion (S. Chen et al, 2001b). In addition, several types of voltage-and G-protein-gated cationic channels can exist as homomeric or heteromeric structures (Hadley et al, 2003;Levitan and Takimoto, 1998;Liao et al, 1996).…”

“…By contrast, homomeric HCN2 channels conduct I h currents with slower kinetics and robust cAMP-evoked shifts in voltage dependence (Ludwig et al, 1998;Robinson and Siegelbaum, 2003). More recently, the formation of heteromeric channels in vitro (Chen et al, 2001b;Er et al, 2003;Much et al, 2003;Proenza et al, 2002;Ulens and Tytgat, 2001;Xue et al, 2002) as well as in vivo (Er et al, 2003;Much et al, 2003) has been described. However, the mechanisms promoting heteromerization have remained unclear.…”

Immunohistochemical localization of Ih channel subunits, HCN1–4, in the rat brain

“…Homomeric channels of HCN4 also show a strong response to cAMP (Ishii et al, 1999;Seifert et al, 1999). In addition, different HCN subunits can coassemble to form functional heteromers (Chen et al, 2001;Ulens and Tytgat, 2001). Expression of a HCN1-HCN2 tandem dimer (Ulens and Tytgat, 2001) or co-injection of recombinant HCN1 and HCN2 cRNAs to Xenopus oocytes (Chen et al, 2001) leads to the formation of I h channels with novel properties that are different from the linear sum of independent populations of HCN1 and HCN2 homomers.…”

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

“…One key residue, R591 of HCN2 located between the PBC helix and β7, interacts directly with the negatively charged exocylic oxygen of the phosphate group. This arginine residue together with its interaction is conserved (Figure 4) and mutation of this residue in CNG, HCN and MloK1 channels decrease the affinity for cyclic nucleotides (Tibbs et al, 1998;Chen et al, 2001;Clayton et al, 2004; Altieri et al, 2008;Harzheim et al, 2008;Schünke et al, 2009). The purine ring interacts through stacking and hydrogen-bond interactions with additional residues located in both the β roll and helix αC.…”