Different Roles for the Cyclic Nucleotide Binding Domain and Amino Terminus in Assembly and Expression of Hyperpolarization-activated, Cyclic Nucleotide-gated Channels

Abstract

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo-or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH 2 -terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.Hyperpolarization-activated "pacemaker" currents (known as I h , I f , or I q and collectively referred to here as I h ) 1 are slowly activating, mixed cationic currents that are important determinants of rhythmic firing in the mammalian heart and brain.The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels that produce I h have been recently cloned (1-5). In addition to I h , cloned HCN channels also produce a time-independent, instantaneous current (I inst ), which is cAMP-sensitive and has a reversal potential similar to that of I h (6). Based on their homology to the tetrameric voltage-gated potassium (K v ) channels and cyclic nucleotide-gated (CNG) channels, HCN channels are thought to be composed of four subunits, each having six transmembrane helices with cytoplasmic amino and carboxyl termini.Four mammalian HCN isoforms (HCN1 to HCN4) have been cloned and heterologously expressed. The time-dependent (I h ) currents produced by these channels differ from each other mainly in their sensitivity to cAMP and rates of activation, with time constants for activation following the series HCN1 Ͻ HCN2 Ͻ HCN3 Ͻ HCN4 (2, 4, 5, 7). Native hyperpolarizationactivated currents in brain and heart differ from the slowly activating currents produced by heterologously expressed homomeric HCN channels, and different subunit isoforms have overlapping expression patterns (8, 9). Thus, it is thought that native pacemaker currents may be produced by a combination of homo-and heteromeric channels. Indeed, the difference in activation rates between I h produced by HCN1 and HCN2 has ...

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).…”

“…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.…”

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

“…Although the molecular compositions of functional cardiac and neuronal I f channels have not been determined, it is clear that the HCN1-4 subunits can generate functional homomeric or heteromeric channels in heterologous expression systems (11)(12)(13)(14). HCN1, for example, co-assembles with HCN2 (11,13) or with HCN4 (14) to form heteromeric channels with properties distinct from each of the homomeric (HCN1, HCN2, or HCN4) channels.…”

“…HCN1, for example, co-assembles with HCN2 (11,13) or with HCN4 (14) to form heteromeric channels with properties distinct from each of the homomeric (HCN1, HCN2, or HCN4) channels. It has also been reported that an HCN2 pore mutant produces dominant-negative suppression of HCN4-encoded (as well as HCN2-encoded) currents in heterologous cells and, in addition, reduces cardiac I f densities (15).…”

Dominant-Negative Suppression of HCN Channels Markedly Reduces the Native Pacemaker Current I f and Undermines Spontaneous Beating of Neonatal Cardiomyocytes

“…By functional interference, it has been proposed that the isoforms HCN1 and HCN2 coassemble to form heteromeric channel complexes like voltage-gated K ϩ channels. 12,13 Very recently, coassembly between these 2 subunits has been shown more directly by use of the yeast 2-hybrid system 14 and a dominant-negative construct. 15 Detailed understanding of the molecular structure and function of the pacemaker channel is critical to any future therapeutic modulation of this current in myocardium.…”