Spontaneous rhythmic activity in mammalian heart and brain depends on pacemaker currents (I h ), which are produced by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here, we report that the mouse HCN2 pacemaker channel isoform also produced a large instantaneous current (I inst(HCN2) ) in addition to the well characterized, slowly activating I h . I inst(HCN2) was specific to expression of HCN2 on the plasma membrane and its amplitude was correlated with that of I h . The two currents had similar reversal potentials, and both were modulated by changes in intracellular Cl

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 ...

Voltage-gated K7 channels (K7.1 to K7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of K7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K7.2, K7.4, and K7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K7 channel inhibitor XE991 (10 M). ML213 (0.1-30M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 M) decreased global intracellular Ca concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 M) caused an increase in the amplitude of whole-cell K7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 M), consistent with ML213 activation of K7 channel currents. Preapplication of XE991 (10 M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K7.4/K7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K7.4- and K7.5-containing channels are essential regulators of DSM excitability and contractility.

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...