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 ...

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH 2 termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast twohybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH 2 terminus of the HCN2 subunit that is responsible for interactions between NH 2 termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH 2 -terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH 2 -terminal determinant of subunit co-assembly and trafficking of pacemaker channels.Pacemaker channels are formed by hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 subunits and are important for generating spontaneous activity in a variety of excitable cells (1, 2). Their primary amino acid sequence predicts a structure similar to those of voltage-gated potassium channels and cyclic nucleotide-gated channels. Thus, HCN subunits are thought to have six transmembrane helices with cytoplasmic amino and carboxyl termini, and to co-assemble as tetramers when forming functional channels. Four mammalian HCN isoforms (HCN1-4) are known (3-5). Co-assembly of different mammalian HCN isoforms has been suggested using electrophysiological analyses (6, 7), and different isoforms have been found in the same cells (8 -12). These findings suggest that the formation of heteromeric channels contributes to the diversity of pacemaker current phenotypes described in vivo.Recently, we suggested that NH 2 -terminal interactions are required for subunit co-assembly and targeting of functional channels to the plasma membrane (13). However, we have not yet determined the region(s) responsible. To identify the critical region, we subdivided the NH 2 terminus based on homology among the different mammalian isoforms of HCN channels. An alignment of NH 2 -terminal amino acid sequences from these isoforms revealed a 52-amino acid domain, which has a high sequence identity (Ͼ90%), and is located immediately adjacent to the first putative transmembrane domain (S1). Using yeast two-hybrid assays, confocal imaging, and patch clamp electrophysiology, we have found that the conserved domain inter...