Functional Characterization of a Trafficking-defective HCN4 Mutation, D553N, Associated with Cardiac Arrhythmia

Ueda, Kazuo; Nakamura, Kazufumi; Hayashi, Takeharu; Inagaki, Natsuko; Takahashi, Megumi; Arimura, Takuro; Morita, Hiroshi; Higashiuesato, Yasushi; Hirano, Yuji; Yasunami, Michio; Takishita, Shuichi; Yamashina, Akira; Ohe, Tohru; Sunamori, Makoto; Hiraoka, Masayasu; Kimura, Akinori

doi:10.1074/jbc.m311953200

Cited by 207 publications

(165 citation statements)

References 39 publications

(41 reference statements)

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“…In particular, the role of HCN channels as the dominant mechanism in heart rate regulation has repeatedly been called into question (4,23). Human genetic studies have suggested that HCN4 channels are important components of the SAN pacemaker machinery (9)(10)(11)(12). A contribution of I f to heart rate determination is further supported by the observation of a heart rate-lowering effect in both humans and rodents when I f is specifically blocked with IVA (13,14,24).…”

Section: Hhcn4 -573x Expression Eliminates Camp Sensitivity Of If Andmentioning

confidence: 69%

“…Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation, the contribution of the major I f -mediating cardiac isoforms HCN2 and HCN4 to SAN function remains highly controversial. Although human genetic (9)(10)(11)(12) and pharmacologic (5,13,14) studies suggest a significant role for HCN4 subunits in SAN pacemaking in humans and rodents, recent data from transgenic mouse models have challenged this view (15,16). Targeted deletion of HCN4 in adult mice was found to cause heart ratedependent sinus pauses but to have no effect on either basal or maximal heart rate or heart rate regulation (16).…”

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confidence: 99%

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Control of heart rate by cAMP sensitivity of HCN channels

Alig

Marger

Mesirca

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

103

114

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''Pacemaker'' f-channels mediating the hyperpolarization-activated nonselective cation current I f are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major I f-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4 -573X expression causes elimination of the cAMP sensitivity of I f and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4 -573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of If, the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMPmediated regulation of If determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4 -573X-linked SAN dysfunction in humans.bradycardia ͉ hyperpolarization cyclic nucleotide-gated ion channels ͉ pacemaker activity ͉ sinoatrial node ͉ transgenic mice H eart automaticity is a fundamental physiological function in higher organisms. The dominant pacemaker of mammalian hearts is the sinoatrial node (SAN). Dysfunction or failure of SAN activity in humans may lead to an abnormally low heart rate, causing such symptoms as palpitations, fatigue, and syncope, which eventually require implantation of a pacemaker device (1). Elevated resting heart rate, in contrast, is associated with increased cardiovascular mortality and morbidity (2, 3). Despite the importance of cardiac pacemaking as a physiological process, the complex mechanisms underlying SAN automaticity and heart rate regulation remain incompletely understood. SAN activity is controlled by the autonomic nervous system; cholinergic and ␤-adrenergic stimulation either slows or accelerates spontaneous SAN activity. Several ionic currents contribute to cardiac automaticity (4-6). Some of these currents are targets of regulation by the autonomic nervous system, but their physiological importance is a matter of debate (4, 5). ''Pacemaker'' f-channels mediating the hyperpolarizationactivated nonselective cation current I f , in particular, are directly regulated by cAMP (7). These channels are homotetrameric or heterotetrameric complexes of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits (8...

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Section: Hhcn4 -573x Expression Eliminates Camp Sensitivity Of If Andmentioning

confidence: 69%

mentioning

confidence: 99%

Control of heart rate by cAMP sensitivity of HCN channels

Alig

Marger

Mesirca

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

103

114

View full text Add to dashboard Cite

show abstract

“…2C) and can affect gating (Johnson and Zagotta, 2005). Also, interestingly, mutations in the C terminus of the HCN4 isoform are known to modify the channel function and to cause arrhythmias (Schulze-Bahr et al, 2003;Ueda et al, 2004;Milanesi et al, 2006). We therefore sought to investigate, first, whether the E515K mutation modifies the biophysical properties of HCN2 channels, Figure 1 A), causing the replacement of glutamic acid 515 by lysine.…”

Section: Resultsmentioning

confidence: 99%

Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy

DiFrancesco¹,

Barbuti²,

Milanesi³

et al. 2011

J. Neurosci.

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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.

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“…Interestingly, three of these mutations are single-point mutations located in the C-terminal fragment containing the C-linker and CNBD domain (22). One of these mutations leads to deletion of the C terminus including CNBD (573X) (23), whereas the other two are single amino acid missense mutations, leading to either reduced channel expression (D553N) or compromised channel function (S672R) (24,25). Corresponding to the loss-of-function phenotype of these mutant HCN4 channels, patients carrying these mutations show symptoms of cardiac arrhythmias or bradycardia (slower heart rate).…”

mentioning

confidence: 99%

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

Xu¹,

Vysotskaya²,

Liu

et al. 2010

Journal of Biological Chemistry

126

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Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å . Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ϳ3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between ␤4 and ␤5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.

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Functional Characterization of a Trafficking-defective HCN4 Mutation, D553N, Associated with Cardiac Arrhythmia

Cited by 207 publications

References 39 publications

Control of heart rate by cAMP sensitivity of HCN channels

Control of heart rate by cAMP sensitivity of HCN channels

Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

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