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The hyperpolarization-activated I(h) current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, I(h) is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance. more...

Ivabradine is a 'heart rate-reducing' agent able to slow heart rate, without complicating side-effects. Its action results from a selective and specific block of pacemaker f-channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f-channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, of which four isoforms (HCN1-4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose-dependent way with half-block concentrations of 0.94 microm for mHCN1 and 2.0 microm for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an 'open-channel' blocker of hHCN4, and a 'closed-channel' blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f-channel kinetics. more...

We found that sinus bradycardia in members of a large family was associated with a mutation in the gene coding for the pacemaker HCN4 ion channel. Pacemaker channels of the sinoatrial node generate spontaneous activity and mediate cyclic AMP (cAMP)-dependent autonomic modulation of the heart rate. The mutation associated with bradycardia is located near the cAMP-binding site; functional analysis found that mutant channels respond normally to cAMP but are activated at more negative voltages than are wild-type channels. These changes, which mimic those of mild vagal stimulation, slow the heart rate by decreasing the inward diastolic current. Thus, diminished function of pacemaker channels is linked to familial bradycardia., (Copyright 2006 Massachusetts Medical Society.) more...

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization. Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity. more...

Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the beta-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-beta-cyclodextrin (MbetaCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of I(f) in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I(f), MbetaCD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4- and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties. more...

'Funny-' (f-) channels of cardiac sino-atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f-channels are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f-channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co-assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co-transfected (HCN4 + HCN1) and concatenated (HCN4-HCN1 (4-1) tandem or HCN1-HCN4 (1-4) tandem) rabbit constructs and compared them with those of the native f-current from rabbit SAN. 4-1 tandem, but not co-transfected, currents had activation kinetics approaching those of If; however, the activation range of 4-1 tandem channels was more negative than that of the f-channel and their cAMP sensitivity were poorer (although that of 1-4 tandem channels was normal). Co-transfection of 4-1 tandem channels with minK-related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f-channels, but a 'context'-dependent mechanism is also likely to modulate the channel properties in native tissues. more...

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating I f currents; miR1OE SAN-like cells instead have only fast-activating I f with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and I f alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate. [Display omitted] • miR-1 increase SAN-specific transcription factors and the number of SAN precursors during sinus node development • Silencing miR-1 impair spontaneous contraction in mESC-derived embryoid bodies. • miR-1 slows the beating rate of pacemaker cells by inhibiting HCN4 translation and thus reducing the I f current • miR-1 Overexpression accelerate I f activation kinetics and decreases cAMP response compatibly with a decreased HCN4/HCN1 [ABSTRACT FROM AUTHOR] more...