HCN4 Expression Research Articles

The Cardiac Conduction System

The human heart beats 2.5 billion times during a normal lifespan, a feat accomplished by cells of the cardiac conduction system (CCS). The functional components of the CCS can be broadly divided into the impulse-generating nodes and the impulse-propagating His-Purkinje system. Human diseases of the conduction system have been identified that alter impulse generation, impulse propagation, or both. CCS dysfunction is primarily due to acquired conditions such as myocardial ischemia/infarct, age-related degeneration, procedural complications, and drug toxicity. Inherited forms of CCS disease are rare, but each new mutation provides invaluable insight into the molecular mechanisms governing CCS development and function. Applying a multidisciplinary approach, which includes human genetic screening, biophysical analysis, and transgenic mouse technology, has yielded a broad array of gene families involved in maintaining normal CCS physiology (Figure 1). In this review, we discuss gene families that have been implicated in human CCS diseases of rhythm, conduction block, accessory conduction, and development (Table). We also investigate evolving therapeutic strategies that may serve as adjuvant or replacement therapy to current implantable pacemakers. Figure 1. Cardiac conduction system cell. Genes identified in human cardiac conduction system disease are highlighted. View this table: Table. Genetic Basis of Conduction System Disease The human sinoatrial node (SAN) is a crescent-shaped, intramural structure with its head located subepicardially at the junction of the right atrium and the superior vena cava and its tail extending 10 to 20 mm along the crista terminalis.26 The SAN has complex 3-dimensional tissue architecture with central and peripheral components made up of distinct ion channel and gap junction expression profiles.27 Central and peripheral cells have different action potential characteristics and conduction properties (Figure 2).27 Experimental and computational models have demonstrated that SAN heterogeneity is necessary to maintain normal automaticity and impulse conduction.28,–,30 Figure 2. Electrophysiological heterogeneity of the …

CSPE Young Investigator Award Session

CSPE Young Investigator Award Session

Molecular architecture of the human specialised atrioventricular conduction axis

The atrioventricular conduction axis, located in the septal component of the atrioventricular junctions, is arguably the most complex structure in the heart. It fulfils a multitude of functions, including the introduction of a delay between atrial and ventricular systole and backup pacemaking. Like any other multifunctional tissue, complexity is a key feature of this specialised tissue in the heart, and this complexity is both anatomical and electrophysiological, with the two being inextricably linked. We used quantitative PCR, histology and immunohistochemistry to analyse the axis from six human subjects. mRNAs for ~ 50 ion and gap junction channels, Ca 2+-handling proteins and markers were measured in the atrial muscle (AM), a transitional area (TA), inferior nodal extension (INE), compact node (CN), penetrating bundle (PB) and ventricular muscle (VM). When compared to the AM, we found a lower expression of Na v1.5, K ir2.1, Cx43 and ANP mRNAs in the CN for example, but a higher expression of HCN1, HCN4, Ca v1.3, Ca v3.1, K ir3.4, Cx40 and Tbx3 mRNAs. Expression of some related proteins was in agreement with the expression of the corresponding mRNAs. There is a complex and heterogeneous pattern of expression of ion and gap junction channels and Ca 2+-handling proteins in the human atrioventricular conduction axis that explains the function of this crucial pathway.

Histological and Biochemical Characteristics in Right Atrium vs. Left Atrium in Patients with Valvular Atrial Fibrillation

Background: It has been known that dominant drivers of atrial fibrillation (AF) exist on left atrium (LA), but the arrhythmogenic foci of ectopic atrial tachycardia commonly exit on right atrium (RA). Therefore, we compared the histologic and biochemical characteristics of LA and RA by analyzing atrial tissues from the patients with valvular AF.Methods and Results: We analyzed immunohistochemical staining of LA appendage tissues and RA appendage tissues taken from 20 patients with valvular AF. The degree of myocardial fibrosis (Sirius-red stain), thickness of subendocardial smooth muscle (SSm) layer (α-SMA stain), and expression of mRNA for multiple biomarkers were quantified.Results: 1. Atrial fibrosis was significantly higher in LA tissues (36.48±12.22% vs. 29.38±9.90%, p<0.0304), and thickness of SSm layer (0.16±0.17 mm vs. 0.01±0.01 mm, p<0.0002) was significantly greater than those in RA tissues. 2. In tissue of LA, relative NCX mRNA expression level was significantly higher (0.012±0.012 vs. 0.005±0.005, p<0.0165), but relative HCN4 mRNA expression level was significantly lower (0.011±0.010 vs. 0.024±0.013, p<0.0123) than those RA tissue.Conclusion: The amount of matrix fibrosis and thickness of SSm layer were greater in LA tissue than in RA tissue. In contrast NCX expression, which is related with triggered activity, was higher in LA, automaticity related HCN4 expression was higher in RA.

Ih “run-up” in rat neocortical neurons and transiently rat or human HCN1-expressing HEK293 cells

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN) are key determinants of CNS functions. Here we describe an increase in hyperpolarization-activated current (I(h)) at the beginning of whole-cell recordings in rat layer 5 cortical neurons. For a closer investigation of this I(h) increase, we overexpressed the predominant layer 5 rat subunit HCN1 in HEK293 cells. We characterized the resulting I(h) in the cell-attached and whole-cell configurations. Breaking into whole-cell configuration led to about a 30% enhancement of rat HCN1-mediated I(h) accompanied by a depolarizing shift in voltage dependence and an accelerated time course of activation. This current enhancement is not species specific; for human HCN1, the current similarly increases in amount and kinetics. Although the changes were bound to cytosolic solution exchange, they were independent of cAMP, ATP, GTP, and the phosphate group donor phosphocreatine. Together, these data provide a characterization of heterologous expression of rat HCN1 and suggest that cytosolic contents suppress I(h). Such a mechanism might constitute a reserve in h-channel function in vivo.

Increased seizure severity and seizure‐related death in mice lacking HCN1 channels

Persistent down-regulation in the expression of the hyperpolarization-activated HCN1 cation channel, a key determinant of intrinsic neuronal excitability, has been observed in febrile seizure, temporal lobe epilepsy, and generalized epilepsy animal models, as well as in patients with epilepsy. However, the role and importance of HCN1 down-regulation for seizure activity is unclear. To address this question we determined the susceptibility of mice with either a general or forebrain-restricted deletion of HCN1 to limbic seizure induction by amygdala kindling or pilocarpine administration. Loss of HCN1 expression in both mouse lines is associated with higher seizure severity and higher seizure-related mortality, independent of the seizure-induction method used. Therefore, down-regulation of HCN1 associated with human epilepsy and rodent models may be a contributing factor in seizure behavior.

Developmental Origin, Growth, and Three-Dimensional Architecture of the Atrioventricular Conduction Axis of the Mouse Heart

The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain. To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2(+/Cre), Mef2c-AHF-Cre, Tbx18(+/Cre)), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. The atrioventricular conduction axis comprises multiple domains with distinctive molecular signatures. The atrial part proliferates from the embryonic atrioventricular canal, along with myocytes derived from the developing atrial septum. The atrioventricular bundle and lower nodal cells are derived from ventricular myocardium.

Expression of key ion channels in the rat cardiac conduction system by laser capture microdissection and quantitative real‐time PCR

The objective of this study was to investigate the molecular basis of the inferior nodal extension (INE) in the atrioventricular junctional area that accounts for arrhythmias. The INE was separated from the adult rat heart by laser capture microdissection. The mRNA expression of ion channels was detected by quantitative real-time PCR. Hierarchical clustering was used to demonstrate clustering of expression of genes in sections. The mRNA expression of HCN4, Ca(v)3.1 and Ca(v)3.2 was high in the INE, atrioventricular node and sino-atrial node, and that of Ca(v)3.2 high in Purkinje fibres. Although the expression of HCN1 and Ca(v)1.3 was low in the rat heart, it was relatively higher in the INE, atrioventricular node and sino-atrial node than in right atrial and right ventricular (working) myocytes. Both HCN2 and Ca(v)1.2 were expressed at higher levels in working myocytes than in nodal tissues and in the INE. Hierarchical clustering analysis demonstrated that the expression of the HCN and calcium channels in INE was similar to that in the slow-response automatic cells and different from that in working myocytes and Purkinje fibres. The expression of HCN and calcium channels in the INE of the adult rat heart is similar to that of slow-response automatic cells and provides a substrate for automatic phase 4 depolarization in cells.

Trafficking and Surface Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Channels in Hippocampal Neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the properties and activity of neurons. However, little is known about the mechanisms that control HCN channel trafficking to subcellular compartments or that regulate their surface expression. Here we studied the dynamics of HCN channel trafficking in hippocampal neurons using dissociated cultures coupled with time lapse imaging of fluorophore-fused HCN channels. HCN1-green fluorescence protein (HCN1-GFP) channels resided in vesicle-like organelles that moved in distinct patterns along neuronal dendrites, and these properties were isoform-specific. HCN1 trafficking required intact actin and tubulin and was rapidly inhibited by activation of either NMDA or AMPA-type ionotropic glutamate receptors in a calcium-dependent manner. Glutamate-induced inhibition of the movement of HCN1-GFP-expressing puncta was associated with increased surface expression of both native and transfected HCN1 channels, and this surface expression was accompanied by augmented I(h). Taken together, the results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of I(h), and hence of neuronal properties, via alterations of HCN1 trafficking and surface expression.

Dynamic changes in HCN2, HCN4, KCNE1, and KCNE2 expression in ventricular cells from acute myocardial infarction rat hearts

Aims: To investigate dynamic changes in the expression of HCN2, HCN4, as well as KCNE1, and KCNE2 mRNA and protein levels in ventricular cells from acute myocardial infarction (AMI) rat hearts. Main methods: An AMI model was induced by ligating the left anterior descending coronary artery (LAD) of Sprague–Dawley rats. The rats were randomly divided into four experimental groups: 24-hour (24 h) post-AMI, 1-week (1w) post-AMI, 2-week (2w) post-AMI, and 4-week (4w) post-AMI; sham-operated control rat groups were established in parallel for each time point. HCN2, HCN4, KCNE1, and KCNE2 mRNA and protein levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and by immunohistochemistry and Western blot, respectively. Key findings: Ventricular arrhythmias occurred in all the post-AMI groups, particularly in the 1w and 2w post-AMI groups. Although HCN2, HCN4, KCNE1, and KCNE2 genes were expressed in the left ventricular myocardium of sham-operated control rats, their expression increased in rat ischemic left ventricular myocardium, with dynamic changes in expression observed 4 weeks after AMI. HCN2, HCN4, and KCNE2 protein levels were highest at 1w and KCNE2 protein levels peaked at 2w post-AMI. Significance: The expression of the HCN2, HCN4, as well as KCNE1, and KCNE2 genes in ventricular cells from AMI rat hearts underwent dynamic changes, reaching peak levels at 1 or 2 weeks post-AMI. The increased expression maybe related to ventricular arrhythmogenesis after AMI.

Mineralocorticoid receptor overexpression in embryonic stem cell-derived cardiomyocytes increases their beating frequency

Cardiac mineralocorticoid receptor (MR) activation triggers adverse cardiovascular events that could be efficiently prevented by mineralocorticoid antagonists. To gain insights into the pathophysiological role of MR function, we established embryonic stem (ES) cell lines from blastocysts of transgenic mice overexpressing the human MR driven by its proximal P1 or distal P2 promoter and presenting with cardiomyopathy, tachycardia, and arrhythmia. Cardiomyocyte differentiation allowed us to investigate the molecular mechanisms contributing to MR-mediated cardiac dysfunction. During cardiac differentiation, wild-type (WT) and recombinant ES cell cultures and excised beating patches expressed endogenous MR along with cardiac gene markers. The two-fold increase in MR protein detected in P1.hMR and P2.hMR cardiomyocytes led to a parallel increase in the spontaneous beating frequency of hMR-overexpressing cardiomyocytes compared with WT. The MR-mediated chronotropic effect was ligand-independent, could be partially repressed by spironolactone, and was accompanied by a significant two- to four-fold increase in mRNA and protein levels of the pacemaker channel HCN1, generating depolarizing If currents, thus revealing a potential new MR target. This was associated with modification in the expression of HCN4, the inward-rectifier potassium channel Kir2.1, and the L-type voltage-dependent calcium channel Cav1.2. We demonstrate that the amplification of MR signalling in ES-derived cardiomyocytes has a major impact on cardiomyocyte contractile properties through an important remodelling of ion channel expression, contributing to arrhythmias. Our results highlight the prominent role of MR function in cardiac physiology and support the benefit of MR antagonists in the management of cardiac dysfunctions.

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

All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface.

Isoform- and Species-Specific Proteolysis of Cardiac Pacemaker Channels

Proteolysis of cardiac pacemaker channels affects biophysical properties of functional channels. Hyperpolarization-activated channels HCN2 and HCN4 can form homomeric or heteromeric functional pacemaker channels in cardiac ventricles. Employing Western blot and immunoprecipitation techniques with antibodies against N- or C- terminus of HCN2 or HCN4, respectively, we investigated protein expression patterns of endogenous HCN2 and HCN4 in cardiac ventricles of small (mouse, rat) and large (sheep, canine) animals and human. Using an antibody against N-terminus of HCN2, more full length protein at 100kD and less cleaved bands around 50kD were detected in small than in large animals. An additional cleaved band around 60kD was exclusively expressed in human. HCN2 C-terminal antibody could not detect any full length protein in all species tested. A 75kD cleaved band was detected in mice, rat, canine and substantially higher in sheep heart ventricles. A 60kD band was observed in human only. Using an N-terminal HCN4 antibody, the full length protein signals (at 160kD) were present in sheep and canine only. The cleaved bands near 100kD predominated in small animals but absent in large animals. With a C-terminal HCN4 antibody, the full length protein was observed in mice, barely detectable in rat, and clearly present in sheep, canine and human. A cleaved band around 100kD predominated in all animals. A minor cleaved band around 50kD appeared in all tested species except human. Overall, there was less HCN2 and more HCN4 proteolysis in small than in large animal cardiac ventricles. Endogenous myocardial HCN2 and HCN4 underwent intensive proteolysis at both N- and C- termini in an isoform- and species-specific pattern. In conclusion, results obtained from HCN2 and HCN4 protein expression in small animals may not be directly applied to large ones including human.

Remodeling of Ion Channel Expression in Patients with Chronic Atrial Fibrillation and Mitral Valvular Heart Disease

Background/AimsUnderlying cardiac pathology and atrial fibrillation (AF) affect the molecular remodeling of ion channels in the atria. Changes in the expression of these molecules have not been demonstrated in Korean patients with mitral valvular heart disease. Thus, the purpose of this study was to analyze ion channel expression in patients with chronic AF and mitral valvular heart disease.MethodsA total of 17 patients (eight males and nine females; mean age, 57 ± 14 years [range, 19 to 77]) undergoing open-heart surgery were included in the study. Twelve patients (seven with coronary artery disease and five with aortic valvular disease) had sinus rhythm, and five patients (all with mitral valvular disease) had chronic, permanent AF. A piece of right atrial appendage tissue (0.5 g) was obtained during surgery. RT-PCR was used to evaluate the expression of L-type Ca2+ channels, ryanodine receptor (RyR2), sarcoplasmic reticular Ca2+-ATPase (SERCA2), gene encoding the rapid component of the delayed rectifier Ikr (HERG), gene encoding calcium-independent transient outward current Ito1 (Kv4.3), gene encoding the ultrarapid component of the delayed rectifier Iku (Kv1.5), K+ channel-interacting protein 2 (KChIP2), hyperpolarization-activated cation channel 2 associated with the pacemaker current If (HCN2), and gene encoding Na+ channel (SCN5A).ResultsReduced L-type Ca2+ channel, RyR2, SERCA2, Kv1.5, and KChIP2 expression and borderline increased HCN2 expression were observed in the patients with AF and mitral valvular heart disease. Left atrial diameter was negatively correlated with RyR2 and KChIP2 expression. Fractional area shortening of the left atrium was positively correlated with RyR2 and KChIP2 expression.ConclusionsAlterations in ion channel expression and the anatomical substrate may favor the initiation and maintenance of AF in patients with mitral valvular heart disease.

Diabetes alters protein expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits in rat nodose ganglion cells

Vagal afferent neurons, serving as the primary afferent limb of the parasympathetic reflex, could be involved in diabetic autonomic neuropathy. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the vagal afferent neurons and play an important role in determining cell membrane excitation. In the present study, the protein expression and the electrophysiological characteristics of HCN channels were investigated in nodose ganglion (NG) afferent neurons (A-fiber and C-fiber neurons) from sham and streptozotocin (STZ)-induced diabetic rats. In the sham NG, HCN1, HCN3, and HCN4 were expressed in the A-fiber neurons; and HCN2, HCN3, and HCN4 were expressed in the C-fiber neurons. Compared to the sham NG neurons, diabetes induced the expression of HCN2 in the A-fiber neurons besides overexpression of HCN1 and HCN3; and enhanced the expression of HCN2 and HCN3 in C-fiber neurons. In addition, whole-cell patch-clamp data revealed diabetes also increased HCN currents in A-fiber and C-fiber neurons. However, we found that diabetes did not alter the total nodose afferent neuron number and the ratio of A-fiber/C-fiber neurons. These results indicate that diabetes induces the overexpression of HCN channels and the electrophysiological changes of HCN currents in the A- and C-fiber nodose neurons, which might contribute to the diabetes-induced alteration of cell excitability in the vagal afferent neurons.

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

In sino-atrial and atrio-ventricular nodal cells, hyperpolarization-activated cyclic nucleotide-gated (HCN) inward current carrying cationic channels, I(f), are expressed that contribute importantly to the diastolic depolarization critical for cardiac pacemaker activity. Although previous studies have demonstrated myocardial expression of both the HCN2 and HCN4 subunits, the specific roles of these subunits in the generation of functional myocardial I(f) channels remain unclear. To explore the molecular compositions of functional cardiac I(f) channels, antibodies targeted against specific C- and N-terminal sequences in HCN2 and HCN4 were exploited to examine HCN2 and HCN4 subunit expression in adult (mouse) heart and to immunoprecipitate endogenous HCN-encoded cardiac I(f) channel complexes. Western blot experiments revealed that although the full-length HCN2 (105 kDa) and HCN4 (160 kDa) proteins are readily detected in transiently transfected HEK-293 cells and in adult (mouse) brain, the molecular mass of the HCN2 protein in the myocardium is approximately 60 kDa. In addition, the myocardial 60-kDa HCN2 protein lacks the C terminus, which contains the cAMP binding domain. In heterologous cells, the C-terminal-truncated HCN2 protein co-assembles with HCN4 to form functional heteromeric HCN channels, which activate faster than homomeric HCN2 or homomeric HCN4 channels, and display properties similar to endogenous myocardial I(f) channels Taken together, these results suggest that functional myocardial I(f) channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit underlies the cAMP-mediated regulation of cardiac I(f) channels.

Activity-dependent regulation of HCN1 protein in cortical neurons

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of I h current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons.

Landmarks and Lineages in the Developing Heart

See related article, pages 1267–1274 One of the overarching concepts underpinning mammalian embryonic development is that of “regulation.” This implies that the lineage potential of a particular cell is broader than its actual fate. Although the influential experiments in this area performed by Hans Driesch in the late 1800s concerned the fate of individual embryonic blastomeres, the concept of regulative development has pervaded all aspects of embryology, overlapping with the concept of regenerative fields. In the context of the heart, a good example is that of frog embryo cells fated to form the dorsal mesocardium and dorsal pericardial (splanchnic) mesoderm but which can “regulate” and form myocardial tissue if the normal heart is injured or extirpated.1 As we delve into the molecular mechanisms of developmental processes, we understand that limitations to cell fate are often set, particularly in the embryo, by geographical or environmental parameters, such as the source and strength of a secreted inductive signal, and these may change with time. Anatomic patterns or landmarks provide vital clues to how we should think about development and indeed disease. For example, segmentation of the embryonic paraxial mesoderm into repetitive units called somites, which bear one of the main progenitor populations for the musculoskeletal system, has been widely studied and it is known that somite segmentation is controlled by a network of synchronized molecular oscillators coupled by the Delta-Notch intercellular signaling system.2 The heart has also long been considered to have a segmental prepattern based, in part, on the series of swellings and constrictions that become evident during early heart tube formation and early fate-mapping experiments suggesting that these were the precursor structures of the chambers and their flanking regions, including the atrioventricular canal (AVC). This notion has been perpetuated as fact in embryology and medical textbooks with …

TRIP8b Splice Variants Form a Family of Auxiliary Subunits that Regulate Gating and Trafficking of HCN Channels in the Brain

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

Effects of KCNE2 on HCN isoforms: distinct modulation of membrane expression and single channel properties

Hyperpolarization-activated cation (HCN) channels give rise to an inward current with similar but not identical characteristics compared with the pacemaker current (I(f)), suggesting that HCN channel function is modulated by regulatory beta-subunits in native tissue. KCNE2 has been proposed to serve as a beta-subunit of HCN channels; however, available data remain contradictory. To further clarify this situation, we therefore analyzed the effect of KCNE2 on whole cell currents, single channel properties, and membrane protein expression of all cardiac HCN isoforms in the CHO cell system. On the whole cell level, current densities of all HCN isoforms were significantly increased by KCNE2 without altering voltage dependence or current reversal. While these results correlated well with the KCNE2-mediated 2.2-fold and 1.6-fold increases of membrane protein levels of HCN2 and HCN4, respectively, no effect of KCNE2 on HCN1 expression was obtained. All HCN subtypes displayed faster activation kinetics upon coexpression with KCNE2. Most importantly, for the first time, we demonstrated modulation of single channel function by KCNE2, thus supporting direct functional interaction with HCN subunits. In the presence of KCNE2, the single channel amplitudes and conductance of HCN1, HCN2, and HCN4 were significantly increased versus control recordings. Mean open time was significantly increased in cells coexpressing HCN2 + KCNE2, whereas it was unaffected in HCN1 + KCNE2 cotransfected cells and reduced in HCN4 + KCNE2 cotransfected cells compared with the respective HCN subunits alone. Thus, we demonstrate KCNE2-mediated distinct effects on HCN membrane expression and direct functional modulation of HCN isoforms, further supporting that KCNE2 surves as a regulatory beta-subunit of HCN channels.