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IV-Spectrin regulates TREK-1 membrane targeting in the heart
Abstract
Cardiac function depends on the highly regulated and coordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K(+) channels have been well-characterized in heart, much less is known about regulation and/or targeting of two-pore K(+) channel (K2P) family members, despite their potential importance in modulation of heart function.Methods and ResultsHere we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K2P channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that βIV-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in heart, and is required for TREK-1 membrane targeting. Mice expressing βIV-spectrin lacking TREK-1 binding (qv(4 J)) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal βIV-spectrin levels in human heart failure.
These data provide new insight into membrane targeting of TREK-1 in heart and establish a broader role for βIV-spectrin in organizing functional membrane domains critical for normal heart function.
... However, this channel is also present in the heart muscle, where it has been less extensively explored. The interaction between spectrin and the TREK-1 channel has been highlighted in a study conducted by Hund et al. in 2014. βIV-spectrin is a structural protein that binds to ankyrin-G, both proteins being strongly involved in the organization of different ion channels in the membrane. ...
... To conclude, it has been shown that βIV-spectrin is required for the correct localization of the TREK-1 channel in the intercalated discs of cardiomyocytes as well as for its arachidonic acid-dependent activity in mice. Also, TREK-1 and βIV-spectrin are physically associated with each other regardless of the binding of βIV-spectrin to ankyrin-G (Hund et al., 2014). Figure 3 βIV-spectrin is involved in the regulation of TREK-1 localization and function in mice heart. ...
... showing an interaction between TREK-1 and βIV-spectrin. Adapted from Hund et al., 2014. B. P11 is required for TASK-1 cellular trafficking TASK-1 is a K2P potassium channel expressed in many cell types, with abundant levels in brain and heart muscle. ...
Two-pore domain (K2P) potassium channels belong to a large family of ion channels implicated in determining and maintaining the resting cell membrane potential. K2P channels are proteins extensively conserved throughout evolution, being present in almost all animal cells. In the nematode Caenorhabditis elegans, 47 genes code K2P channels sub-units, but only three of them have been characterized and reported in the literature. By tagging a certain number of them with fluorescent proteins (CRISPR/Cas9), we have found that nine channels are co-expressed in body wall muscle, showing a highly specific sub-cellular distribution. The most fascinating distribution was the one of TWK-28, which exhibits a polarized comet-like pattern that occupy only the anterior tip of each body wall muscle cell. In order to elucidate the cellular mechanisms underlying this particular distribution, we performed a genetic screen on the novel TWK-28 gain-of-function strain. We revealed that genes belonging to Dystrophin-Associated Protein Complex (DAPC) are involved in determining the amount of this channel at the muscle cell surface. DAPC is composed of at least 10 intra and extracellular proteins and plays a key role in physically connecting the extracellular matrix to the actin cytoskeleton. Interestingly, when tagging multiple components of DAPC with fluorescent proteins by CRISPR/Cas9 gene editing, we found that most of the dystrophin-associated proteins, such as syntrophin/STN-1, dystrobrevin/DYB-1 or even sarcoglycans (SGCA-1 and SGCB-1), show a particularly asymmetric distribution in muscle. We also revealed the, to date excluded, presence of dystroglycan/DGN-1 in body wall muscle of C. elegans. Finally, the asymmetric distribution of TWK-28 along the antero-posterior axis on a cellular and tissue scale, suggests that the Planar Cell Polarity pathways might be implicated. By gene candidate approach of the WNT pathway, we showed that proteins such as Disheveled, ROR/CAM-1 or WNT ligand/EGL-20 can modify the localization of TWK-28 by driving it into a new posterior sub-complex in the muscle cells
... In the rat heart, Kcnk2 mRNA and protein expression has been described in both atrial and ventricular tissue samples (Table 2) [28,29,32,33,149]. However, in the mouse heart, most studies describe ventricular-dominant K2P2.1 (TREK-1) expression or mRNA abundance patterns [16,26,41]. Abundant K2P2.1 (TREK-1) expression was also detected in the porcine heart, with the highest expression levels in the sinoatrial and atrioventricular nodal tissue [36,37] and in human cardiac tissue samples, where again ventricular dominant K2P2.1 (TREK-1) expression could be observed [10,37,40,41]. ...
... In the murine and the rat heart, KCNK3 mRNA was detected, both in atrial as well as in ventricular tissue samples (Northern blot, RT-PCR, Taq-Man qPCR; Table 2) [15,16,18,25,26,34,44,45,47]. Humans, however, show an almost atrial-specific K2P3.1 (TASK-1) expression within the heart with 14-to 16-fold lower expression levels in ventricular tissue (RT-PCR, Taq-Man qPCR, microarray, bulk RNAseq, Western blot) [10,12,14,39,40,49,54,56,57]. ...
... Switching the pH level from pH 7.4 to 7.8, however, resulted in significant prolongation of atrial effective refractory periods [49]. Global Kcnk3 knockout mice exhibited a phenotype of QTc prolongation (around 30%), prolongation of single cell APDs or monophasic action potentials and a broad QRS complex [25,26]. In transgenic Kcnk3 knockout rats, APD prolongation as well as resting membrane depolarization was described [163]. ...
Two-pore-domain potassium (K2P-) channels conduct outward K+ currents that maintain the resting membrane potential and modulate action potential repolarization. Members of the K2P channel family are widely expressed among different human cell types and organs where they were shown to regulate important physiological processes. Their functional activity is controlled by a broad variety of different stimuli, like pH level, temperature, and mechanical stress but also by the presence of lipids or pharmacological agents. In patients suffering from cardiovascular diseases, alterations in K2P-channel expression and function have been observed, suggesting functional significance and a potential therapeutic role of these ion channels. For example, upregulation of atrial specific K2P3.1 (TASK-1) currents in atrial fibrillation (AF) patients was shown to contribute to atrial action potential duration shortening, a key feature of AF-associated atrial electrical remodelling. Therefore, targeting K2P3.1 (TASK-1) channels might constitute an intriguing strategy for AF treatment. Further, mechanoactive K2P2.1 (TREK-1) currents have been implicated in the development of cardiac hypertrophy, cardiac fibrosis and heart failure. Cardiovascular expression of other K2P channels has been described, functional evidence in cardiac tissue however remains sparse. In the present review, expression, function, and regulation of cardiovascular K2P channels are summarized and compared among different species. Remodelling patterns, observed in disease models are discussed and compared to findings from clinical patients to assess the therapeutic potential of K2P channels.
... Spectrins are important for membrane integrity and ultrastructure (8). Recent studies 23 demonstrate novel roles for IV -spectrin in regulating heart function through the organization of 24 local signaling domains (9)(10)(11). Specifically, β IV -spectrin targets CaMKII to membrane substrates 25 at the intercalated disc, a specialized cardiac 'synapse'-like structure important for intercellular 26 mechanical and electrical communication. Importantly, our group and others have identified 27 significant alterations in spectrins and spectrin-based pathways in human HF and animal models 28 of HF, although the functional consequences are unknown and untested (9)(10)(11)(12)(13)(14)(15)(16). ...
... Specifically, β IV -spectrin targets CaMKII to membrane substrates 25 at the intercalated disc, a specialized cardiac 'synapse'-like structure important for intercellular 26 mechanical and electrical communication. Importantly, our group and others have identified 27 significant alterations in spectrins and spectrin-based pathways in human HF and animal models 28 of HF, although the functional consequences are unknown and untested (9)(10)(11)(12)(13)(14)(15)(16). Based on 29 previous work showing an important role for CaMKII in modulating hypertrophy and HF in 30 response to chronic stress (4-6, 12, 17), we hypothesized that IV -spectrin may serve as a novel 31 therapeutic target for abrogating adverse cardiac remodeling and HF. ...
... ostensibly due to stress-induced downregulation of IV -spectrin (10,26). Furthermore, targeted 23 ablation of IV -spectrin/CaMKII interaction in vivo (qv 3J allele) prevents STAT3 dysregulation and 24 preserves cardiac function in response to chronic pressure overload. ...
Heart failure (HF) remains a major source of morbidity and mortality in the U.S. The multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII) has emerged as a critical regulator of cardiac hypertrophy and failure, although the mechanisms remain unclear. Previous studies have established that the cytoskeletal protein βIV-spectrin coordinates local CaMKII signaling. Here we sought to determine the role of a spectrin/CaMKII complex in maladaptive remodeling in HF. Chronic pressure overload (6 weeks transaortic constriction, TAC) induced a decrease in cardiac function in WT mice but not in animals expressing truncated βIV-spectrin lacking spectrin/CaMKII interaction (qv3J). Underlying observed differences in function was an unexpected differential regulation of STAT3-related genes in qv3J TAC hearts. In vitro experiments demonstrate that βIV-spectrin serves as a target for CaMKII phosphorylation, which regulates its stability. Cardiac-specific βIV-spectrin knockout (βIV-cKO) mice show STAT3 dysregulation, fibrosis and decreased cardiac function at baseline similar to WT TAC. STAT3 inhibition restored normal cardiac structure and function in βIV-cKO and WT TAC hearts. Our studies identify a novel spectrin-based complex essential for regulation of the cardiac response to chronic pressure overload. We anticipate that strategies targeting the new spectrin-based "statosome" will be effective at suppressing maladaptive remodeling in response to chronic stress.
... In contrast, α I -and α II -spectrin along with β I -spectrin account for major spectrin components at the lateral membrane. Finally, α II -, β II -, β IV -spectrin are principal family members localized to intercalated disc (Figure 1) [5,7,[11][12][13][14]. The localization of spectrin isoforms to myocyte membrane domains important for cell-cell communication (e.g. ...
... In contrast, β IV -spectrin is highly localized with ankyrin-G at the cardiac intercalated disc, a specialized membrane domain important for electrical and mechanical cell-to-cell coupling [7,[40][41][42]. While it is clear that ankyrin-G is required for proper localization of β IV -spectrin and intercalated disc proteins, including Na v 1.5 (primary cardiac Na v ), the role of β IV -spectrin in ion channel targeting is less established [12,[40][41][42][43]. It is likely that β IV -spectrin, in fact, plays a more prominent role in regulation of Na v 1.5 rather than targeting (discussed in more detail below). ...
... It is likely that β IV -spectrin, in fact, plays a more prominent role in regulation of Na v 1.5 rather than targeting (discussed in more detail below). However, β IV -spectrin has been linked to membrane targeting of other cardiac ion channels, specifically, the two-pore K + channel, TREK-1 [12,44]. TREK-1 channels encoded by KCNK2, belong to the two-poredomain background potassium channel protein family (K 2P ) and are expressed in nervous and cardiovascular systems where they regulate cell membrane excitability and participate in transduction of a variety of environmental stimuli [45,46]. ...
Introduction:
In the heart, pathways that transduce extracellular environmental cues (e.g. mechanical force, inflammatory stress) into electrical and/or chemical signals at the cellular level are critical for the organ-level response to chronic biomechanical/neurohumoral stress. Specifically, a diverse array of membrane-bound receptors and stretch-activated proteins converge on a network of intracellular signaling cascades that control gene expression, protein translation, degradation and/or regulation. These cellular reprogramming events ultimately lead to changes in cell excitability, growth, proliferation, and/or survival. Areas covered: The actin/spectrin cytoskeleton has emerged as having important roles in not only providing structural support for organelle function but also in serving as a signaling "superhighway," linking signaling events at/near the membrane to distal cellular domains (e.g. nucleus, mitochondria). Furthermore, recent work suggests that the integrity of the actin/spectrin cytoskeleton is critical for canonical signaling of pathways involved in cellular response to stress. This review discusses these emerging roles for spectrin and consider implications for heart function and disease. Expert Commentary: Despite growth in our understanding of the broader roles for spectrins in cardiac myocytes and other metazoan cells, there remain important unanswered questions, the answers to which may point the way to new therapies for human cardiac disease patients.
... The SAK candidate TREK-1 is expressed in the heart of rodents Fink et al., 1996;Putzke et al., 2007;Tan et al., 2002;Terrenoire et al., 2001;Unudurthi et al., 2016;Xian Tao et al., 2006) and recent data also show expression in the human heart (Hund et al., 2014). However, the other members of the mechanosensitive K 2P channel subfamily (Fig. 2), namely TREK-2 and TRAAK, are only weakly or not expressed in rodent as well as in human heart (Bang et al., 2000;Fink et al., 1998;Gu et al., 2002;Kim et al., 2001a;Lesage and Lazdunski, 2000;Lesage et al., 2000a;Meadows et al., 2001). ...
... However, the other members of the mechanosensitive K 2P channel subfamily (Fig. 2), namely TREK-2 and TRAAK, are only weakly or not expressed in rodent as well as in human heart (Bang et al., 2000;Fink et al., 1998;Gu et al., 2002;Kim et al., 2001a;Lesage and Lazdunski, 2000;Lesage et al., 2000a;Meadows et al., 2001). In contrast, other reports show a strong cardiac expression of TREK-2 and TRAAK in humans (Hund et al., 2014;Ozaita and Vega-Saenz de Miera, 2002). In rat, TREK-1 mRNA expression was reported in the atria, as well as in left, right and septal ventricular myocytes (Aimond et al., 2000;Liu and Saint, 2004;Tan et al., 2004;Terrenoire et al., 2001). ...
... However, TREK-1 channels interact with the actin cytoskeleton, involving the intracellular C-terminus (Lauritzen et al., 2005). Recent studies suggest that this interaction might be also mediated by b IV spectrin (Hund et al., 2014). The actin cytoskeleton binding however antagonizes the stretch-sensitivity of TREK-1 (Lauritzen et al., 2005). ...
This review focuses on the role and the molecular candidates for the cardiac stretch-activated potassium current (SAK). The functional properties of two-pore domain potassium (K2P) channel TREK-1, a major candidate for the cardiac SAK are analyzed and the molecular mechanism of stretch-activation in K2P potassium channels is discussed. Furthermore, the functional modulation of TREK-1 by different cardiac interaction partners, as well as evidence for the functional role of the stretch-dependent TREK-1 and its putative subunits in the heart is reviewed. In addition, we summarize the recent evidence that TREK-1 is involved in the pathogenesis of human cardiac arrhythmias.
... TREK-1 is highly expressed in the nervous system, where it regulates depression phenotypes and nociception. Expression of TREK-1 has also been identified in heart with proposed roles in regulation of myocyte membrane excitability, [7][8][9][10][11][12][13][14] although its role in specific cardiac phenotypes remains unclear. ...
... The cDNA for b IV -spectrin and TREK-1 (C-terminal cytoplasmic region 305-422) fusion proteins was cloned from a human heart cDNA library, as described previously. 8 Constructs for in vitro translation and fusion protein expression were generated by engineering cDNAs in frame into pcDNA3.1 + (Invitrogen) and pGEX6P1 (GE Healthcare). ...
... Experimental mice were generated by crossing these animals with mice expressing Cre under the cardiac promoter a-myosin heavy chain (aMHC-Cre), resulting in cardiac-specific deletion of TREK-1 (aMHC-Kcnk2 f/f ). The qv 4J mice (which express a mutant b IV -spectrin allele with a premature stop codon in spectrin repeat 10) 8,17 were obtained from Jackson Laboratory (Bar Harbor, ME). All experiments were performed in male mice aged 2 months (30 wild-type [WT], 30 aMHC-Kcnk2 f/f , and 12 qv 4J animals were used for studies). ...
Background
Two‐pore K⁺ channels have emerged as potential targets to selectively regulate cardiac cell membrane excitability; however, lack of specific inhibitors and relevant animal models has impeded the effort to understand the role of 2‐pore K⁺ channels in the heart and their potential as a therapeutic target. The objective of this study was to determine the role of mechanosensitive 2‐pore K⁺ channel family member TREK‐1 in control of cardiac excitability.
Methods and Results
Cardiac‐specific TREK‐1–deficient mice (αMHC‐Kcnk f/f) were generated and found to have a prevalent sinoatrial phenotype characterized by bradycardia with frequent episodes of sinus pause following stress. Action potential measurements from isolated αMHC‐Kcnk2 f/f sinoatrial node cells demonstrated decreased background K⁺ current and abnormal sinoatrial cell membrane excitability. To identify novel pathways for regulating TREK‐1 activity and sinoatrial node excitability, mice expressing a truncated allele of the TREK‐1–associated cytoskeletal protein βIV‐spectrin (qv 4J mice) were analyzed and found to display defects in cell electrophysiology as well as loss of normal TREK‐1 membrane localization. Finally, the βIV‐spectrin/TREK‐1 complex was found to be downregulated in the right atrium from a canine model of sinoatrial node dysfunction and in human cardiac disease.
Conclusions
These findings identify a TREK‐1–dependent pathway essential for normal sinoatrial node cell excitability that serves as a potential target for selectively regulating sinoatrial node cell function.
... This editorial refers to 'Re-trafficking of hERG reverses long QT syndrome 2 phenotype in human iPS-derived cardiomyocytes' by A. Mehta et al., this issue. This editorial refers to 'b IV -Spectrin regulates TREK-1 membrane targeting in the heart' by T.J. Hund et al., 2014;102: 166 -175. Normal cardiac electrical activity depends on the proper membrane expression of a number of ion channels in specialized domains of the sarcolemma of cardiac myocytes. ...
... Two recent articles in Cardiovascular Research bring new building blocks to the emerging scheme of molecular cardiac electrophysiology. 2,3 The article of Hund et al. 2 reports a new role of the actin-associated protein, b IV -spectrin. In cardiac myocytes, this protein regulates the targeting of ion channels into the intercalated disc by binding to the adapter protein ankyrin-G. ...
... Two recent articles in Cardiovascular Research bring new building blocks to the emerging scheme of molecular cardiac electrophysiology. 2,3 The article of Hund et al. 2 reports a new role of the actin-associated protein, b IV -spectrin. In cardiac myocytes, this protein regulates the targeting of ion channels into the intercalated disc by binding to the adapter protein ankyrin-G. ...
... A number of studies on zebrafish and mice that inhibited plasma membrane trafficking of TREK-1 by inactivating the interacting proteins POPDC1 and POPDC2 revealed exerciseand age-dependent sick sinus syndrome and atrioventricular block (78,86), suggesting a role for TREK-1 in cardiac automaticity. Similarly, transgenic overexpression of a Cterminal truncation of beta IV spectrin, which also disrupts TREK-1 plasma membrane trafficking, results in sick sinus syndrome (87). These studies provide indirect evidence of TREK-1-mediated effects on SAN automaticity. ...
... The authors determined that TREK-1 protein is indeed expressed in both murine and rabbit SAN, and TREK-1like background currents were reduced in patch-clamped SAN cells isolated from cardiac-specific TREK-1 KO mice (αMHC-Kcnk2 f/f ). Also, freely moving, telemetered αMHC-Kcnk2 f/f mice exhibited sinus bradycardia at rest, consistent with studies by Hund et al. (87) where disrupted plasma membrane TREK-1 trafficking induced sick sinus syndrome. Paradoxically, isolated TREK-1 KO SAN cells exhibited increased rather than decreased firing rates as compared with wildtype (WT) SAN. ...
The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.
... Adult (2-4 months) C57BL/6J male and female wildtype (WT) and truncated βIV-spectrin (qv 4J ) littermate mice were used. The qv 4J mice were obtained from Jackson Laboratory and express a Sptnb4 allele with a spontaneous insertion point mutation at C4234T (Q1358→Stop) resulting in a premature stop codon proximal to the STAT3 binding region in β IV -spectrin (53,54). periostin MerCreMer mice (55) were cross-bred with β IV -spectrin floxed mice (12) to obtain tamoxifeninducible β IV -ifKO mice. ...
... Primary ventricular cardiac fibroblast lysates were prepared by washing cells in PBS and scraping in PhosphoSafe™ Extraction Buffer (Millipore 71296) supplemented with protease inhibitor cocktail (Millipore P8340) and analyzed using SDS-PAGE and immunoblotting, as described (10,12,54). Briefly, equal protein loading was achieved using standard BCA protein assay protocols and verified by Ponceau staining of immunoblots. ...
Fibrosis is a pronounced feature of heart disease and the result of dysregulated activation of resident cardiac fibroblasts (CFs). Recent work identified stress-induced degradation of the cytoskeletal protein βIV-spectrin as an important step in CF activation and cardiac fibrosis. Further, loss of βIV-spectrin was found to depend on Ca²⁺/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of βIV-spectrin and CF activity. Computational screening and mass spectrometry revealed a critical serine residue (S2250 in mouse, S2254 in human) in βIV-spectrin phosphorylated by CaMKII. Disruption of βIV-spectrin/CaMKII interaction or alanine substitution of βIV-spectrin Ser2250 (βIV-S2250A) prevented CaMKII-induced degradation, while a phospho-mimetic construct (βIV-S2254E) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous βIV-S2254A and βIV-S2254E constructs in βIV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared to wild-type (WT) CFs. βIV-S2254A but not βIV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiologic targeting of βIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II (AngII), resulting in increased proliferation and STAT3 nuclear accumulation. While therapeutic delivery of exogenous WT βIV-spectrin partially reversed these trends, βIV-S2254A completely negated increased CF proliferation and STAT3 translocation. Moreover, we observed βIV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure (HF). Together, these data illustrate a considerable role for the βIV-spectrin/CaMKII interaction in activating profibrotic signaling.
... There is growing appreciation for the multifunctional nature of spectrin family members beyond providing structural support for the cell membrane. For example, spectrins support longrange cellular communication involving, in part, coordination of signaling nanodomains for ion channels [42,[53][54][55][56][57][58][59][60]. More recently, it has been discovered that spectrins modulate gene expression to control remodeling of cell function in response to chronic stress stimuli, although the precise mechanisms remain to be determined [61][62][63]. ...
... TREK-1 function has been well characterized in the nervous system with identified roles in nociception and depression phenotypes [79]. The channel is also expressed in heart across species, including mice and human [57,[80][81][82][83]. Interestingly, β IV -spectrin is required for normal TREK-1 membrane expression in SAN and ventricular myocytes. ...
The cardiac conduction system is an extended network of excitable tissue tasked with generation and propagation of electrical impulses to signal coordinated contraction of the heart. The fidelity of this system depends on the proper spatio-temporal regulation of ion channels in myocytes throughout the conduction system. Importantly, inherited or acquired defects in a wide class of ion channels has been linked to dysfunction at various stages of the conduction system resulting in life-threatening cardiac arrhythmia. There is growing appreciation of the role that adapter and cytoskeletal proteins play in organizing ion channel macromolecular complexes critical for proper function of the cardiac conduction system. In particular, members of the ankyrin and spectrin families have emerged as important nodes for normal expression and regulation of ion channels in myocytes throughout the conduction system. Human variants impacting ankyrin/spectrin function give rise to a broad constellation of cardiac arrhythmias. Furthermore, chronic neurohumoral and biomechanical stress promotes ankyrin/spectrin loss of function that likely contributes to conduction disturbances in the setting of acquired cardiac disease. Collectively, this review seeks to bring attention to the significance of these cytoskeletal players and emphasize the potential therapeutic role they represent in a myriad of cardiac disease states.
... In the heart ßIV-spectrin is localized in myocyte intercalated discs and has a major role in the organization of ion channels like cardiac voltage-gated sodium channels, Na v 1.5 or potassium channels, like TREK-1 (10). ßIV-spectrin mutant mice have shown impairments in cardiac repolarization and arrhythmia (9). ...
... A mouse model with ßIV-spectrin mutant mice has shown impaired cardiac function through different mechanisms, involving ßIV-spectrin (10,11). Myocytes of SPTBN4 mutant mice have shown highly eccentric myocyte growth (11). ...
ßIV-spectrin is a protein of the spectrin family which is involved in the organization of the cytoskeleton structure and is found in high quantity in the axon initial segment and the nodes of Ranvier. Together with ankyrin G, ßIV-spectrin is responsible for the clustering of KCNQ2/3-potassium channels and NaV-sodium channels. Loss or reduction of ßIV-spectrin causes a destabilization of the cytoskeleton and an impairment in the generation of the action potential, which leads to neuronal degeneration. Furthermore, ßIV-spectrin has been described to play an important role in the maintenance of the neuronal polarity and of the diffusion barrier. ßIV-spectrin is also located in the heart where it takes an important part in the structural organization of ion channels and has also been described to participate in cell signaling pathways through binding of transcription factors. We describe two patients with a severe form of ßIV-spectrin deficiency. Whole-exome sequencing revealed the homozygous stop mutation c.6016C>T (p.R2006*) in the SPTBN4 gene. The phenotype of these patients is characterized by profound psychomotor developmental arrest, respiratory insufficiency and deafness. Additionally one of the patients presents with cardiomyopathy, optical nerve atrophy, and mitochondrial dysfunction. This is the first report of a severe form of ßIV-spectrin deficiency with hypertrophic cardiomyopathy and mitochondrial dysfunction.
... Recently, several loss-of-function variants in β IV -spectrin have been identified in human patients with neuropathy, myopathy, and congenital deafness (34,35). We anticipate that our findings will have important implications for human patients with both acquired and inherited forms of disease, given: (a) conservation of the β IV -spectrin/STAT3 complex across mouse and humans; (b) our previous studies showing loss of β IV -spectrin in human heart failure (36); and (c) results from this study showing transcriptional changes in human fibroblasts with acute spectrin knockdown (Supplemental Figure 5). ...
... Adult (2-4 months) C57BL/6J male and female wildtype (WT, control) and truncated β IV -spectrin (qv 4J ) littermate mice were used. The qv 4J mice were obtained from Jackson Laboratory and express a Sptnb4 allele with a spontaneous insertion point mutation at C4234T (Q1358→Stop) resulting in a premature stop codon proximal to the STAT3 binding region in β IV -spectrin (21,36). periostin MerCreMer mice (22) were cross-bred with β IV -spectrin floxed mice (16) to obtain tamoxifen-inducible β IV -ifKO mice. ...
Increased fibrosis is a characteristic remodeling response to biomechanical and neurohumoral stress and a determinant of cardiac mechanical and electrical dysfunction in disease. Stress-induced activation of cardiac fibroblasts (CF) is a critical step in the fibrotic response, although the precise sequence of events underlying activation of these critical cells in vivo remain unclear. Here, we test the hypothesis that a βIV-spectrin/STAT3 complex is essential for maintenance of a quiescent phenotype (basal non-activated state) in CFs. We report increased fibrosis, decreased cardiac function, and electrical impulse conduction defects in genetic and acquired mouse models of βIV-spectrin deficiency. Loss of betaIV-spectrin function promotes STAT3 nuclear accumulation and transcriptional activity, altered gene expression and CF activation. Furthermore, we demonstrate that a quiescent phenotype may be restored in βIV-spectrin deficient fibroblasts by expressing a βIV-spectrin fragment including the STAT3-binding domain or through pharmacological STAT3 inhibition. We find that in vivo STAT3 inhibition abrogates fibrosis and cardiac dysfunction in the setting of global βIV-spectrin deficiency. Finally, we demonstrate that fibroblast-specific deletion of βIV-spectrin is sufficient to induce fibrosis and decreased cardiac function. We propose that the βIV-spectrin/STAT3 complex is a determinant of fibroblast phenotype and fibrosis, with implications for remodeling response in cardiovascular disease.
... Among K + channels, modest upregulation of Kcnj2 and Kcnj11 (both in the t-tubule GO category) was observed along with higher upregulation of Kcna4 (Kv1.4; 1.50-fold increase), which is expressed in t-tubules and sarcolemma [28] , and Kcnk2 (TREK-1; 1.65x), which is expressed in intercalated discs [29] . Among Ca 2+ channels, Cacna1s (Cav1.1; ...
... It mediates transient outward currents during the initial phase of depolarization [37] and might therefore counteract the lengthening of the action potential expected with a reduction in NBCe1 activity [38] . TREK-1 is expressed in intercalated discs [29] and plays an important role in regulating excitability of the sinoatrial node [39] . Thus, increased expression and activity of TREK-1 has the potential to counteract the electrical effects of the loss of NBCe1 activity in intercalated discs. ...
AIM
To investigate the hypothesis that cardiomyocyte-specific loss of the electrogenic NBCe1 Na⁺-HCO3⁻ cotransporter is cardioprotective during in vivo ischemia-reperfusion (IR) injury.
METHODS
An NBCe1 (Slc4a4 gene) conditional knockout mouse (KO) model was prepared by gene targeting. Cardiovascular performance of wildtype (WT) and cardiac-specific NBCe1 KO mice was analyzed by intraventricular pressure measurements, and changes in cardiac gene expression were determined by RNA Seq analysis. Response to in vivo IR injury was analyzed after 30 min occlusion of the left anterior descending artery followed by 3 h of reperfusion.
RESULTS
Loss of NBCe1 in cardiac myocytes did not impair cardiac contractility or relaxation under basal conditions or in response to β-adrenergic stimulation, and caused only limited changes in gene expression patterns, such as those for electrical excitability. However, following ischemia and reperfusion, KO heart sections exhibited significantly fewer apoptotic nuclei than WT sections.
CONCLUSION
These studies indicate that cardiac-specific loss of NBCe1 does not impair cardiovascular performance, causes only minimal changes in gene expression patterns, and protects against IR injury in vivo .
... TREK-1 interacts with POPDC and β (IV) spectrin. These 2 proteins promote TREK-1 membrane trafficking and increase its membrane expression (Hund, Snyder et al. 2014, Schindler, Scotton et al. 2016. TREK-1 can also be activated by heat and a wide range of volatile and gaseous anesthetics , Gruss, Bushell et al. 2004). ...
... β(IV)-Spectrin plays a key role in membrane trafficking of TREK-1 in the cardiomyocytes: loss of β(IV)-Spectrin activity results in a decrease of TREK-1 expression. This was associated with heart failure, arrhythmias and a long QT syndrome(Hund et al 2014). Under stress conditions, TREK-1 is important for heart automaticity. ...
In humans, most cardiovascular disorders lead to the destruction of cardiac tissue which will be replaced by fibrosis, leading to arrhythmia and reduced contractile function, resulting in an increase in ventricular load. In order to maintain an overall cardiac output, cardiomyocytes undergo hypertrophic response, leading to pathological hypertrophy and heart failure. This increase in ventricular load, have to be sensed by mechanosensors such as the mechanosensitive ion channels such as TREK-1. Unlike mammals, adult zebrafish (zf) can fully regenerate their heart after an extensive insult through cardiomyocyte dedifferentiation followed by proliferation. We believe that in adult mammals, cardiomyocyte proliferation has been blocked/inhibited. Therefore it’s likely that genes which respond to increased ventricular load in mammals and trigger pathological hypertrophy will trigger cardiomyocyte proliferation during heart regeneration in zf. In this study we show that zTREK1a and zTREK1b have similar biophysical and pharmacological properties to mammalian TREK1 and they are important for successful zebrafish heart regeneration.
... Beyond, ankyrin-G and CaMKII, βIV spectrin regulates TREK-1, a mechano-sensitive K 2P channel (Heurteaux et al., 2004). More specifically, βIV spectrin and TREK-1 associate, and βIV spectrin is required for TREK-1 localization and function in ventricular myocytes (Hund et al., 2014). Mice lacking TREK-1 binding display delayed action potential repolarization and arrhythmia. ...
... These data support a broader role for βIV spectrin in cardiovascular electrophysiology by regulation of TREK-1 membrane localization and function. Like βII spectrin, βIV spectrin levels are altered in human heart failure as well as in animal models of cardiovascular disease (Hund et al., 2014). ...
Ankyrins are adaptor proteins critical for the expression and targeting of cardiac membrane proteins, signaling molecules, and cytoskeletal elements. Findings in humans and animal models have highlighted the in vivo roles for ankyrins in normal physiology and in cardiovascular disease, most notably in cardiac arrhythmia. For example, human ANK2 loss-of-function variants are associated with a complex array of electrical and structural phenotypes now termed “ankyrin-B syndrome,” whereas alterations in the ankyrin-G pathway for Nav channel targeting are associated with human Brugada syndrome. Further, both ankyrin-G and -B are now linked with acquired forms of cardiovascular disease including myocardial infarction and atrial fibrillation. Spectrins are ankyrin-associated proteins and recent studies support the critical role of ankyrin-spectrin interactions in normal cardiac physiology as well as regulation of key ion channel and signaling complexes. This review will highlight the roles of ankyrins and spectrins in cardiovascular physiology as well as illustrate the link between the dysfunction in ankyrin- and spectrin-based pathways and disease.
... The TWIK-related potassium channel-1 (Trek1) is located in epithelial cells, 9 endothelial cells and the cardiovascular system, 10 and has also been identified in the intestine. 11 Recent reports have suggested that Trek1 regulates barrier function. ...
... The allergic mice were treated with SIT following published procedures with a minor modification. Briefly, an increasing dose of OVA (the specific antigen) was administered to the mice by gavage in 0.3 ml saline daily for 14 days as follows: 10 mg (days 1 and 2), 50 mg (days 3 and 4), 0.1 mg (days 5-7), 0.25 mg (days 8 and 9) and 0.5 mg (days [10][11][12][13][14]. ...
The disruption of epithelial barrier integrity is an important factor in the pathogenesis of various immune disorders. However, the restitution of the compromised barrier functions is difficult. This study investigates the regulation of TWIK-related potassium channel-1 (Trek1) in the restitution of intestinal epithelial barrier functions. The human colon epithelial cell line T84 was cultured in monolayers and used to observe epithelial barrier functions in vitro. An intestinal allergy mouse model was created. Cytokine levels were determined by enzyme-linked immunosorbent assay and western blotting. The results showed that Trek1 deficiency induced T84 monolayer barrier disruption. Allergic responses markedly suppressed the expression of Trek1 in the intestinal epithelia via activating the mitogen-activated protein kinase pathways and increasing the expression of histone deacetylase-1. The inhibition of histone deacetylase-1 by sodium butyrate or the administration of a butyrate-producing probiotic (Clostridium butyricum) restored the intestinal epithelial barrier functions and markedly enhanced the effect of antigen-specific immunotherapy. The data suggest that Trek1 is required for the maintenance of intestinal epithelial barrier integrity. Allergic responses induce an insufficiency of Trek1 expression in the intestinal epithelia. Trek1 expression facilitates the restoration of intestinal epithelial barrier functions in an allergic environment.Cellular & Molecular Immunology advance online publication, 16 February 2015; doi:10.1038/cmi.2014.137.
... This current component has not been adequately described so far in cardiac ventricular muscle. TREK-1 channels are known to be expressed in human atria and ventricles [15], and their expression is higher in subendocardial as compared to subepicardial myocytes [52,68]. TREK-1 channels are stretch sensitive and may play a role in mechano-electric feedback in the heart [24]. ...
... TREK-1 channels are highly expressed in rodent and human cardiac muscle cells [10,15,24,52], and single TREK-1 channels can easily be recorded from rat atrial or ventricular cardiomyocytes [19,24,56]. However, the outward current carried by TREK-1 channels (I TREK ) is difficult to characterise because there are so many different potassium channels in cardiomyocytes. ...
We studied the potassium current flowing through TREK-1 channels in rat cardiac ventricular myocytes. We separated the TREK-1 current from other current components by blocking most other channels with a blocker cocktail. We tried to inhibit the TREK-1 current by activating protein kinase A (PKA) with a mixture of forskolin and isobutyl-methylxanthine (IBMX). Activation of PKA blocked an outwardly rectifying current component at membrane potentials positive to -40 mV. At 37 °C, application of forskolin plus IBMX reduced the steady-state outward current measured at positive voltages by about 52 %. Application of the potassium channel blockers quinidine or tetrahexylammonium also reduced the steady-state outward current by about 50 %. Taken together, our results suggest that the increase in temperature from 22 to 37 °C increased the TREK-1 current by a factor of at least 5 and that the average density of the TREK-1 current in rat cardiomyocytes at 37 °C is about 1.5 pA/pF at +30 mV. The contribution of TREK-1 to the action potential was assessed by using a dynamic patch clamp technique. After subtraction of simulated TREK-1 currents, action potential duration at 50 or 90 % repolarisation was increased by about 12 %, indicating that TREK-1 may be functionally important in rat ventricular muscle. During sympathetic stimulation, inhibition of TREK-1 channels via PKA is expected to prolong the action potential primarily in subendocardial myocytes; this may decrease the transmural dispersion of repolarisation and thus may serve to prevent the occurrence of arrhythmias.
... Immobilized glutathione-S-transferase-fusion proteins were incubated with 100 μg left ventricular heart lysate overnight in pull-down buffer at 4°C. 19 The samples were washed 3× in pull-down buffer, eluted, and proteins were separated by SDS/PAGE (sodium dodecyl sulfate/polyacrylamide gel electrophoresis). The gels were transferred to nitrocellulose and immunoblotted. ...
... For these experiments, we used a β IV spectrin mutant mouse model (qv 4J mice) harboring a premature stop codon in the 10th spectrin repeat, resulting in a truncated polypeptide lacking ankyrin-G-binding activity (located in 15th spectrin repeat; see Figure 4A). 18,19 As expected, a glutathione-S-transferase-β IV spectrin fusion protein harboring the qv 4J mutation lacked binding activity for ankyrin-G and Na v 1.5 ( Figure 4B). In contrast to findings in neurons, 27 qv 4J myocytes displayed no significant difference in ankyrin-G or Na v channel expression compared with control hearts by immunoblot ( Figure 4C and 4D). ...
Rationale:
Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined.
Objective:
Define the in vivo mechanisms underlying Nav1.5 membrane regulation.
Methods and results:
Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G(-/-) mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin-dependent kinase II to the intercalated disc. Mechanistically, βIV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin-dependent kinase II-δ; however, βIV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation.
Conclusions:
Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.
... TREK-1-deficient PCs showed significantly increased spontaneous firing rate, diastolic depolarization rate, and action potential duration (APD) at 50% repolarization, suggesting that TREK-1 is essential for PC excitability. Furthermore, several studies reported that disrupting TREK-1 plasma membrane trafficking results in SAN dysfunction [38,41]. These data illustrated that the mechanoactivated ion channel TREK-1 contributes to SAN automaticity and its abnormality can induce SND (Fig. 1). ...
Purpose of Review
The sinoatrial node (SAN), the natural pacemaker of the heart, is responsible for generating electrical impulses and initiating each heartbeat. Sinoatrial node dysfunction (SND) causes various arrhythmias such as sinus arrest, SAN block, and tachycardia/bradycardia syndrome. Unraveling the underlying mechanisms of SND is of paramount importance in the pursuit of developing effective therapeutic strategies for patients with SND. This review provides a concise summary of the most recent progress in the signaling regulation of SND.
Recent Findings
Recent studies indicate that SND can be caused by abnormal intercellular and intracellular signaling, various forms of heart failure (HF), and diabetes. These discoveries provide novel insights into the underlying mechanisms SND, advancing our understanding of its pathogenesis.
Summary
SND can cause severe cardiac arrhythmias associated with syncope and an increased risk of sudden death. In addition to ion channels, the SAN is susceptible to the influence of various signalings including Hippo, AMP-activated protein kinase (AMPK), mechanical force, and natriuretic peptide receptors. New cellular and molecular mechanisms related to SND are also deciphered in systemic diseases such as HF and diabetes. Progress in these studies contributes to the development of potential therapeutics for SND.
... TREK-1 channels also have a potential role in regulating the normal activity of sinoatrial node-hosted pacemakers by preventing the occurrence of ventricular extrasystoles (55, 70). Inhibition of TREK-1 channels via PKA during sympathetic stimulation may decrease transmural dispersion of repolarization and prevent the occurrence of arrhythmias (58), indicating that TREK-1 may have an essential function in the cardiac conduction system (71). In cardiomyocytes, the refractory period is critical in preventing premature excitation and arrhythmias. ...
Mechano-electric feedback is one of the most important subsystems operating in the cardiovascular system, but the underlying molecular mechanism remains rather unknown. Several proteins have been proposed to explain the molecular mechanism of mechano-transduction. Transient receptor potential (TRP) and Piezo channels appear to be the most important candidates to constitute the molecular mechanism behind of the inward current in response to a mechanical stimulus. However, the inhibitory/regulatory processes involving potassium channels that operate on the cardiac system are less well known. TWIK-Related potassium (TREK) channels have emerged as strong candidates due to their capacity for the regulation of the flow of potassium in response to mechanical stimuli. Current data strongly suggest that TREK channels play a role as mechano-transducers in different components of the cardiovascular system, not only at central (heart) but also at peripheral (vascular) level. In this context, this review summarizes and highlights the main existing evidence connecting this important subfamily of potassium channels with the cardiac mechano-transduction process, discussing molecular and biophysical aspects of such a connection.
... Adult (2-4 mos, 18-22 g) male and female C57BL/6J wildtype (WT, control) and β IV -spectrin truncated (qv 4J ) littermate mice were used (see Table 1 for complete list of abbreviations). qv 4J animals genetically express a Spnb4 allele with a spontaneous insertion point mutation at C4234T (Q1358 > Stop) resulting in a premature stop codon in β IV -spectrin repeat 10 leading to the lack of repeats 11 through the C-terminus including the putative STAT3 binding region [22,24,25]. qv 4J animals were acquired from Jackson Laboratory. ...
Cardiac fibroblasts (CFs) maintain the fibrous extracellular matrix (ECM) that supports proper cardiac function. Cardiac injury induces a transition in the activity of CFs to promote cardiac fibrosis. CFs play a critical role in sensing local injury signals and coordinating the organ level response through paracrine communication to distal cells. However, the mechanisms by which CFs engage cell-cell communication networks in response to stress remain unknown. We tested a role for the action-associated cytoskeletal protein βIV-spectrin in regulating CF paracrine signaling. Conditioned culture media (CCM) was collected from WT and βIV-spectrin deficient (qv4J) CFs. WT CFs treated with qv4J CCM showed increased proliferation and collagen gel compaction compared to control. Consistent with the functional measurements, qv4J CCM contained higher levels of pro-inflammatory and pro-fibrotic cytokines and increased concentration of small extracellular vesicles (30–150 nm diameter, exosomes). Treatment of WT CFs with exosomes isolated from qv4J CCM induced a similar phenotypic change as that observed with complete CCM. Treatment of qv4J CFs with an inhibitor of the βIV-spectrin-associated transcription factor, STAT3, decreased the levels of both cytokines and exosomes in conditioned media. This study expands the role of the βIV-spectrin/STAT3 complex in stress-induced regulation of CF paracrine signaling.
... AC9 interacts with all three POPDC isoforms, as shown using a host of cellular and biochemical methods. POPDC proteins are localized in multiple membrane compartments in adult cardiomyocytes including the intercalated disk and the lateral plasma membrane, where AC9 and TREK-1 also reside (Hund et al, 2014;Brand & Schindler, 2017;Li et al, 2019). POPDC1 can homodimerize, involving residues within the Popeye domain and likely the transmembrane domain (Kawaguchi et al, 2008;Russ et al, 2011). ...
The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the β-adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.
... In ventricular myocytes, αII and βII spectrin are found at the transversetubule and sarcoplasmic reticulum membranes 8 , whereas βIV spectrin is located at the intercalated disc 30 . The three spectrins have been reported to play important roles in cardiovascular electrophysiology 12,30,31,32 . However, cardiomyocyte-selective αII spectrin-de cient mice display cardiomyocyte hypertrophy and cardiac brosis 31 . ...
βII spectrin is a cytoskeletal protein known to be tightly linked to heart development and cardiovascular electrophysiology. However, roles of βII spectrin in cardiac contractile function and post-myocardial infarction pathological remodeling remain unclear. Here, we uncovered that the levels of serum βII spectrin breakdown products (βII SBDPs) were significantly increased in patients with acute myocardial infarction. Consistently, βII spectrin was degraded into βII SBDPs by calpain in mouse hearts after ischemia/reperfusion (I/R) injury. Cardiac-specific βII spectrin deletion results in spontaneous development of cardiac contractile dysfunction, cardiac hypertrophy and fibrosis. Moreover, deletion of βII spectrin in the adult heart exacerbated I/R-induced cardiomyocyte death and heart failure, while restoration of βII spectrin expression by adenoviral saRNA delivery in the heart reduced I/R injury. IP–LC–MS/MS and functional studies revealed that βII spectrin is indispensable for mitochondrial complex I activity and respiratory function. Mechanistically, βII spectrin interacted with mitochondrial complex I to mediate its assembly by crosslinking with actin filaments (F-actin) to maintain F-actin stability. These findings identify βII spectrin as an essential mitochondrial cytoskeletal element for preserving mitochondrial homeostasis and cardiac function.
... However, these animals suffered from exercise-induced sick sinus syndrome. A similar phenotype was observed when trafficking of the channel to the plasma membrane was impaired by deletion of the TREK-1 interacting partners βIV spectrin or the Popeye-domain containing proteins POPDC1 and POPDC2 [142,143]. Furthermore, POPDC1 and POPDC2 double knockout animals exhibited signs of atrioventricular conduction disorder (AV block). The mutations disrupting the regulation of TREK-1 by POPDC1 or POPDC2 proteins lead to the development of familial AV block [144,145]. ...
The two-pore domain K2P subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K2P subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K+ current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K2P channels, and focus on the physiological roles of mechanically induced hyperpolarization.
... Also, endothelin-1 (ET-1) was discovered as a TREK1 upstream regulator in calciummediated vasocontraction via Gq protein-coupled protein kinase C (PKC) signaling [108]. β(IV) spectrin, an actin-associated protein, was found to regulate TREK1 membrane trafficking by colocalization with the channel [109,110]. Therefore, TREK1 channels might coordinate with G protein coupled receptors and its downstream effector proteins to regulate myocardial functions under physiological conditions. On the other hand, histone deacetylase (HDAC) inhibitors increased TREK1 expression and prolonged action potential duration in murine atrial cardiomyocytes, indicating that epigenetic changes also modulate TREK1 functions [111]. ...
The family of two-pore domain potassium (K2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K2P channels leads to heterogeneous influences on human diseases. The role of individual K2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K2P channels.
... βII-spectrin deficiency in the heart also causes sinus node dysfunction, cytoskeletal remodeling, ryanodine receptor mislocalization, arrhythmias and more rapid progression to heart failure in response to increased afterload [40]. Beyond βII-spectrin, βIV-spectrin has been shown to regulate the targeting of STAT3 to the ICD in response to pressure overload [77] and has reduced expression during heart failure in humans [78]. ...
The non-contractile cytoskeleton in cardiomyocytes is comprised of cytoplasmic actin, microtubules, and intermediate filaments. In addition to providing mechanical support to these cells, these structures are important effectors of tension-sensing and signal transduction and also provide networks for the transport of proteins and organelles. The majority of our knowledge on the function and structure of these cytoskeletal networks comes from research on proliferative cell types. However, in recent years, researchers have begun to show that there are important cardiomyocyte-specific functions of the cytoskeleton. Here we will discuss the current state of cytoskeletal biology in cardiomyocytes, as well as research from other cell types, that together suggest there is a wealth of knowledge on cardiac health and disease waiting to be uncovered through exploration of the complex signaling networks of cardiomyocyte non-sarcomeric cytoskeletal proteins.
... Additionally, spontaneous murine mutations arising in β IV -spectrin known as quivering (qv) mice have revealed a critical role of β IV -spectrins (6). For instance, the qv 4J mutation disrupts the interaction between β IV -spectrin and ankyrin-G, thereby mislocalizing the two-pore potassium channel TREK1, which causes cardiac arrhythmias in mice (7). Likewise, qv 3J mice lacking the C-terminal CaMKII-β IV -spectrin interaction develop disorganized and dysfunctional membranes in neuronal, pancreatic (8), and cardiac muscle cells (3). ...
βIV-Spectrin, along with ankyrin and Ca2+/calmodulin-dependent kinase II (CaMKII), has been shown to form local signaling domains at the intercalated disc, while playing a key role in the regulation of Na+ and K+ channels in cardiomyocytes. In this issue of the JCI, Unudurthi et al. show that under chronic pressure overload conditions, CaMKII activation leads to βIV-spectrin degradation, resulting in the release of sequestered STAT3 from the intercalated discs. This in turn leads to dysregulation of STAT3-mediated gene transcription, maladaptive remodeling, fibrosis, and decreased cardiac function. Overall, this study presents interesting findings regarding the role of CaMKII and βIV-spectrin under physiological as well as pathological conditions.
... Two K2P subfamily members, TWIK related acid sensitive potassium channel (TASK) and TWIK related potassium channel (TREK) channels are highly expressed in human cardiac tissue (3) and implicated in cardiac arrhythmogenesis (4). TREK channels, which are present in atrial (5) and ventricular tissue (6,7), have been of significant interest because of their role in cardiac chronotropic function (8), dynamic modulation by adrenergic signaling (9), and biomechanical stretch (10). Recent work has identified K2P channels as significant modulators of Drosophila diastolic function (11). ...
Cardiac two pore domain potassium channels (K2P) exist in organisms from Drosophila to humans, however their role in cardiac function is not known. We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK related potassium channel (TREK-1 or Kcnk2), exhibit exaggerated pressure overload induced concentric hypertrophy and alterations in fetal gene expression, yet retain preserved systolic and diastolic cardiac function. While cardiomyocyte specific deletion of TREK-1 in response to in vivo pressure overload resulted in cardiac dysfunction, TREK-1 deletion in fibroblasts prevented deterioration in cardiac function. The absence of pressure overload induced dysfunction in TREK-1 KO mice was associated with diminished cardiac fibrosis and reduced activation of c-Jun N-terminal kinase activity (JNK) in cardiomyocytes and fibroblasts. These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload-induced cardiac dysfunction and serve as a conceptual basis for its inhibition for as a potential therapy.
... Immunoblot analysis. Ventricular lysates were analyzed by SDS-PAGE and immunoblotting, as described elsewhere (19,22,23,29). Equal protein loading was achieved using standard BCA protocols and verified by Ponceau staining of immunoblots. ...
The mechanisms underlying CaMKII-induced arrhythmias in ischemia/reperfusion (I/R) are not fully understood. In this study, we tested the hypothesis that CaMKII increases late Na+ current (INa,L) via phosphorylation of Nav1.5 at Ser571 during I/R, thereby increasing arrhythmia susceptibility. To test our hypothesis, isolated, Langendorff-perfused hearts from wildtype (WT) mice and mice expressing Nav channel variants Nav1.5-Ser571E (S571E) and Nav1.5-Ser571A (S571A) were studied. Wildtype (WT) hearts showed a significant increase in the levels of phosphorylated CaMKII and Nav1.5 at Ser571 [(p-Nav1.5(S571)] following 15 minutes of global ischemia (just before onset of reperfusion). Optical mapping studies revealed an increase in action potential duration (APD) and APD dispersion without changes in conduction velocity during I/R in WT and S571E hearts compared to S571A. At the same time, WT and S571E hearts showed an increase in spontaneous arrhythmia events (e.g. premature ventricular contractions) and an increase in inducibility of reentrant arrhythmias during reperfusion. Pretreatment of WT hearts with Na+ channel blocker mexiletine (10 μM) normalized APD dispersion and reduced arrhythmia susceptibility during I/R. We conclude that CaMKII-dependent phosphorylation of Nav1.5 is a crucial driver for increased INa,L, arrhythmia triggers and substrate during I/R. Selective targeting of this CaMKII-dependent pathway may have therapeutic potential for reducing arrhythmias in the setting of I/R.
... The screen identified both α and β spectrin as both interacting partner with the first 24 amino acids of the C terminal region. Both spectrin subtypes are membrane cytoskeleton components [23,24] and there is a growing body of literature implicating spectrins in the clustering of ion channels (e.g., sodium and potassium channels) at specific subcellular loci [25][26][27], often indirectly via specific ankyrins [22,28,29]. We confirmed the spectrin interactions via co-immunoprecipitation from mouse brain tissue and also identified ankyrin B as part of the putative Cav3.2/spectrin binding complex. ...
This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/β) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/β) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/β) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.
Electronic supplementary material
The online version of this article (10.1186/s13041-018-0368-5) contains supplementary material, which is available to authorized users.
... However, it hasn't been found (yet) in human heart. 103,104 Lastly, it must be noted that Kir channels and Nav1.5 are, although mainly activated by other stimuli, also mechanically affected-at least in human embryonic kidney cells (reviewed in Ref. 103). ...
This review presents an extensively integrated model of the cardiac intercalated disc (ID), a highly orchestrated structure that connects adjacent cardiomyocytes. Classically, three main structures are distinguished: gap junctions (GJs) metabolically and electrically connect cytoplasm of adjacent cardiomyocytes; adherens junctions (AJs) connect the actin cytoskeleton of adjacent cells; and desmosomes function as cell anchors and connect intermediate filaments. Furthermore, ion channels reside in the ID. Mutations in ID proteins have been associated with cardiac arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. However, rather than being independent, all ID components work together intensively by multifunctional proteins such as ZO-1, Ankyrin G and β-catenin, integrating mechanical and electrical functions. GJs form a plaque surrounded by the perinexus in which free connexons reside; the connexome integrates NaV channels, the desmosome and GJs; and the area composita hosts AJs and desmosomes, also integrated as adhering junctions. Furthermore, the transitional junction connects sarcomeres to the plasma membrane. Lastly, this review integrates all these findings in comprehensible figures, illustrating the interdependencies of ID proteins.
... Interestingly, TREK-1 in myocytes interacts with β IV -spectrin and ankyrin G [66], which are involved in membrane targeting and complex formation of a number of cardiac ion channels [67]. A cardiac-specific knockout of Kcnk2 encoding TREK-1 displays a stress-induced sinus bradycardia similar to the one observed in Popdc1 and Popdc2 null mutants [68], which means that the sinus bradycardia phenotype of POPDC mutants could at least be partially explained by the loss of the POPDC-TREK-1 interaction. ...
The Popeye domain containing (POPDC) genes encode a novel class of cAMP effector proteins, which are abundantly expressed in heart and skeletal muscle. Here we will review their role in striated muscle as deduced from work in cell and animal models and the recent analysis of patients carrying a missense mutation in POPDC1. Evidence suggests that POPDC proteins control membrane trafficking of interacting proteins. Furthermore, we will discuss the current catalogue of established protein-protein interactions. In recent years, the number of POPDC-interacting proteins is rising and currently includes ion channels (TREK-1), sarcolemma-associated proteins serving functions in mechanical stability (Dystrophin), compartmentalization (Caveolin 3), scaffolding (ZO-1), trafficking (NDRG4, VAMP2/3) and repair (Dysferlin), or acting as a guanine nucleotide exchange factor for Rho-family GTPases (GEFT). Recent evidence suggests that POPDC proteins might also control the cellular level of the nuclear proto-oncoprotein c-Myc. These data suggests that this family of cAMP-binding proteins probably serves multiple roles in striated muscle.
... Second, TREK-1 has a distinct C terminus binding site with microtubule associated protein (Mtap2) in neurons, resulting in an enhanced membrane TREK-1 expression and functional currents (Sandoz et al., 2008). Third, β IV -spectrin, an actin-associated protein, is required for the membrane targeting and activity of TREK-1 in the cardiomyocytes (Hund et al., 2014). It remains to be determined whether any of these mechanisms exist in astrocytes. ...
We have recently shown that a linear current-to-voltage (I-V) relationship of membrane conductance (passive conductance) reflects the intrinsic property of K+ channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (VM) essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K+ channels remains elusive. TREK-1 two-pore domain K+ channel (K2P) is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of this heterodimer channel to the membrane and contributing to astrocytes’ passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K+ channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, VM, or membrane input resistance (Rin) in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K+ channels may shed light on this long-standing and important question in astrocyte physiology.
... Importantly, also TREK-1 is involved in cytoskeletal arrangement (Lauritzen et al., 2005). Moreover, the actin-associated protein ßIV-spectrin regulates TREK-1 membrane targeting (Hund et al., 2014) and so do Popdc proteins (Froese et al., 2012). Therefore, a complex regulatory network involving cytoskeletal components, mechanosensors, and Popdc proteins exist, Fig. 1. ...
Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which have been accumulated since its discovery about 15 years ago. We will be focusing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for heart and skeletal muscle.
... Beta 1e3 spectrins are overall similar in sequence and domain organization , although beta-4 spectrin has additional sequence between the final spectrin repeat and the PH domain (Berghs et al., 2000). Beta-4 spectrin also associates with calmodulin-dependent protein kinase 2 (CAM kinase 2) through this sequence and recruits CAM kinase 2 to cardiac intercalated discs and axon initial segments (Hund et al., 2010Hund et al., , 2014). The phenotypes of beta-spectrin knockout mice indicate that beta-2 spectrin is the generally expressed isoform with nonredundant functions, while beta-1 spectrin is essential for erythrocyte survival and beta-4 spectrin has specialized roles in neurons and cardiomyocytes. ...
Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that early axon initial segments and epithelial lateral membranes initially were based on spectrin-ankyrin-cell adhesion molecule assemblies and subsequently served as "incubators," where ion transporters independently acquired ankyrin-binding activity through positive selection.
... Ventricular myocytes were isolated from Langendorff-perfused adult mouse hearts, as described previously. 22,26,31 I Na recordings were performed on freshly isolated (<1 h in culture) myocytes at room temperature (20-22 °C) by a conventional whole-cell patch- clamp technique with an Axon 200B patch-clamp amplifier controlled by a personal computer using a Digidata 1320A acquisition board and the pClamp 10.3 software (Axon Instruments). I Na,L was measured using two different methods: 1) average current over the interval 50-150 ms following the peak current; or 2) integral over the same time interval. ...
-Voltage-gated Na(+) channels (Nav) are essential for myocyte membrane excitability and cardiac function. Nav current (INa) is a large amplitude, short duration "spike" generated by rapid channel activation followed immediately by inactivation. However, even under normal conditions, a small "late" component of INa (INa,L) persists due to incomplete/failed inactivation of a subpopulation of channels. Notably, INa,L is directly linked with both congenital and acquired disease states. The multifunctional Ca(2+)/calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of INa,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Nav1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of INa,L in vivo remains unknown.
-To determine the in vivo role of Ser571, two Scn5a knock-in mouse models were generated expressing either: 1) Nav1.5 with a phosphomimetic mutation at Ser571 (S571E), or 2) Nav1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates INa,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in INa,L promote abnormal repolarization and intracellular Ca(2+) handling, and increase susceptibility to arrhythmia at the cellular and animal level. Importantly, Ser571 is required for maladaptive remodeling and arrhythmias in response to pressure overload.
-Our data provide the first in vivo evidence for the molecular mechanism underlying CaMKII activation of the pathogenic INa,L. Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Nav1.5 specifically tunes INa,L without altering critical physiological components of the current.
... Expression of TREK-1 has recently been demonstrated in the human heart. 61 However, it is currently unclear to what extent I Kss contributes to the repolarization of the human atrium. Moreover, it is not clear whether these TREK-like currents are atrial selective. ...
Aims
Noradrenaline plays an important role in the modulation of atrial electrophysiology. However, the identity of the modulated channels, their mechanisms of modulation, and their role in the action potential remain unclear. This study aimed to investigate the noradrenergic modulation of an atrial steady-state outward current (IKss).
Methods and results
Rat atrial myocyte whole-cell currents were recorded at 36°C. Noradrenaline potently inhibited IKss (IC50 = 0.90 nM, 42.1 ± 4.3% at 1 µM, n = 7) and potentiated the L-type Ca2+ current (ICaL, EC50 = 136 nM, 205 ± 40% at 1 µM, n = 6). Noradrenaline-sensitive IKss was weakly voltage-dependent, time-independent, and potentiated by the arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (EYTA; 10 µM), or by osmotically induced membrane stretch. Noise analysis revealed a unitary conductance of 8.4 ± 0.42 pS (n = 8). The biophysical/pharmacological properties of IKss indicate a TREK-like K+ channel. The effect of noradrenaline on IKss was abolished by combined β1-/β2-adrenoceptor antagonism (1 µM propranolol or 10 µM β1-selective atenolol and 100 nM β2-selective ICI-118,551 in combination), but not by β1- or β2-antagonist alone. The action of noradrenaline could be mimicked by β2-agonists (zinterol and fenoterol) in the presence of β1-antagonist. The action of noradrenaline on IKss, but not on ICaL, was abolished by pertussis toxin (PTX) treatment. The action of noradrenaline on ICaL was mediated by β1-adrenoceptors via a PTX-insensitive pathway. Noradrenaline prolonged APD30 by 52 ± 19% (n = 5; P < 0.05), and this effect was abolished by combined β1-/β2-antagonism, but not by atenolol alone.
Conclusion
Noradrenaline inhibits a rat atrial TREK-like K+ channel current via a PTX-sensitive mechanism involving co-operativity of β1-/β2-adrenoceptors that contributes to atrial APD prolongation.
Spectrin, as one of the major components of a plasma membrane-associated cytoskeleton, is a cytoskeletal protein composed of the modular structure of α and β subunits. The spectrin-based skeleton is essential for preserving the integrity and mechanical characteristics of the cell membrane. Moreover, spectrin regulates a variety of cell processes including cell apoptosis, cell adhesion, cell spreading, and cell cycle. Dysfunction of spectrins is implicated in various human diseases including hemolytic anemia, neurodegenerative diseases, ataxia, heart diseases, and cancers. Here, we briefly discuss spectrins function as well as the clinical manifestations and currently known molecular mechanisms of human diseases related to spectrins, highlighting that strategies for targeting regulation of spectrins function may provide new avenues for therapeutic intervention for these diseases.
The spontaneous activity of the sinoatrial node initiates the heartbeat. Sino-atrial node dysfunction (SND) and sick sinoatrial (sick sinus) syndrome are caused by the heart's inability to generate a normal sinoatrial node action potential. In clinical practice, SND is generally considered an age-related pathology, secondary to degenerative fibrosis of the heart pacemaker tissue. However, other forms of SND exist, including idiopathic primary SND, which is genetic, and forms that are secondary to cardiovascular or systemic disease. The incidence of SND in the general population is expected to increase over the next half century, boosting the need to implant electronic pacemakers. During the last two decades, our knowledge of sino-atrial node physiology and of the pathophysiological mechanisms underlying SND has advanced considerably. This review summarizes the current knowledge about SND mechanisms and discusses the possibility of introducing new pharmacologic therapies for treating SND.
The spontaneous activity of the sinoatrial node initiates the heartbeat. Sinoatrial node dysfunction (SND) and sick sinoatrial syndrome are caused by the heart's inability to generate a normal sinoatrial node action potential. In clinical practice, SND is generally considered an age-related pathology, secondary to degenerative fibrosis of the heart pacemaker tissue. However, other forms of SND exist, including idiopathic primary SND, which is genetic, and forms that are secondary to cardiovascular or systemic disease. The incidence of SND in the general population is expected to increase over the next half century, boosting the need to implant electronic pacemakers. During the last two decades, our knowledge of sinoatrial node physiology and of the pathophysiological mechanisms underlying SND has advanced considerably. This review summarizes the current knowledge about SND mechanisms and discusses the possibility of introducing new pharmacologic therapies for treating SND.
Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 61 is January 7, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
TWIK-related K⁺ channel (TREK-1) two-pore-domain potassium (K2P) channels mediate background potassium currents and regulate cellular excitability in many different types of cells. Their functional activity is controlled by a broad variety of different physiological stimuli, such as temperature, extracellular or intracellular pH, lipids and mechanical stress.
By linking cellular excitability to mechanical stress, TREK-1 currents might be important to mediate parts of the mechanoelectrical feedback described in the heart. Furthermore, TREK-1 currents might contribute to the dysregulation of excitability in the heart in pathophysiological situations, such as those caused by abnormal stretch or ischaemia-associated cell swelling, thereby contributing to arrhythmogenesis.
In this review, we focus on the functional role of TREK-1 in the heart and its putative contribution to cardiac mechanoelectrical coupling. Its cardiac expression among different species is discussed, alongside with functional evidence for TREK-1 currents in cardiomyocytes. In addition, evidence for the involvement of TREK-1 currents in different cardiac arrhythmias, such as atrial fibrillation or ventricular tachycardia, is summarized. Furthermore, the role of TREK-1 and its interaction partners in the regulation of the cardiac heart rate is reviewed. Finally, we focus on the significance of TREK-1 in the development of cardiac hypertrophy, cardiac fibrosis and heart failure.
βII spectrin is critical for integrating membrane and cytoskeletal domains in excitable and non-excitable cells. The role of βII spectrin for vertebrate function is illustrated by dysfunction of βII spectrin-based pathways in disease. Recently, defects in βII spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of βII spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that βII spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, βII spectrin levels were decreased in atrial samples of patients with atrial fibrillation compared with tissue from patients in sinus rhythm. Further, compared with left ventricular samples from non-failing hearts, βII spectrin levels were significantly decreased in left ventricle of ischemic- and non-ischemic heart failure patients. Left ventricle samples of canine and murine heart failure models confirm reduced βII spectrin protein levels. Mechanistically, we identify that βII spectrin levels are tightly regulated by post-translational mechanisms, namely calcium- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca(2+)- and calpain-dependent loss of βII spectrin downstream effector proteins including ankyrin-B in heart. In summary, our findings illustrate that βII spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca(2+)- and calpain-dependent proteolysis.
Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.
Ankyrins (ankyrin-R, -B, and -G) are adapter proteins linked with defects in metazoan physiology. Ankyrin-B (encoded by ANK2) loss-of-function mutations are directly associated with human cardiovascular phenotypes including sinus node disease, atrial
fibrillation, ventricular tachycardia, and sudden cardiac death. Despite the link between ankyrin-B dysfunction and monogenic
disease, there are no data linking ankyrin-B regulation with common forms of human heart failure. Here, we report that ankyrin-B
levels are altered in both ischemic and non-ischemic human heart failure. Mechanistically, we demonstrate that cardiac ankyrin-B
levels are tightly regulated downstream of reactive oxygen species, intracellular calcium, and the calcium-dependent protease
calpain, all hallmarks of human myocardial injury and heart failure. Surprisingly, βII-spectrin, previously thought to mediate ankyrin-dependent modulation in the nervous system and heart, is not coordinately
regulated with ankyrin-B or its downstream partners. Finally, our data implicate ankyrin-B expression as required for vertebrate
myocardial protection as hearts deficient in ankyrin-B show increased cardiac damage and impaired function relative to wild-type
mouse hearts following ischemia reperfusion. In summary, our findings provide the data of ankyrin-B regulation in human heart
failure, provide insight into candidate pathways for ankyrin-B regulation in acquired human cardiovascular disease, and surprisingly,
implicate ankyrin-B as a molecular component for cardioprotection following ischemia.
Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.
TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model.
Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na(+) channels underlie cellular excitability and control neuronal communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na(+) channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β(IV)-spectrin as a multifunctional regulatory platform for Na(+) channels in mice. We found that β(IV)-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β(IV)-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na(+) channels, via direct phosphorylation by β(IV)-spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.
Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes,
epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including
ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify
a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal KATP channels directly associate with ankyrin-B in heart via the KATP channel α-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression,
membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating KATP channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with KATP channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively,
our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.
Ion channel reorganization is a critical step in the pro-arrhythmogenic remodelling process that occurs in heart disease. Ankyrin-B (AnkB) is required for targeting and stabilizing ion channels, exchangers, and pumps. Despite a wealth of knowledge implicating the importance of AnkB in human cardiovascular physiology, nothing is known regarding the role of AnkB in common forms of acquired human disease.
We present the first report of AnkB regulation following myocardial infarction (MI). AnkB protein levels were reduced in the infarct border zone 5 days following coronary artery occlusion in the canine. We also observed a dramatic increase in AnkB mRNA levels 5 days post-occlusion. Surprisingly, the expression of the upstream AnkB cytoskeletal component beta2-spectrin was unchanged in post-infarct tissues. However, protein levels and/or membrane expression of downstream AnkB-associated ion channels and transporters Na+/K+ ATPase, Na+/Ca2+ exchanger, and IP3 receptor were altered 5 days post-occlusion. Interestingly, protein levels of the protein phosphatase 2A, an AnkB-associated signalling protein, were significantly affected 5 days post-occlusion. AnkB and PP2A protein levels recovered by 14 days post-occlusion, whereas Na+/K+ ATPase levels recovered by 2 months post-occlusion.
These findings reveal the first evidence of ankyrin remodelling following MI and suggest an unexpected divergence point for regulation between ankyrin and the underlying cytoskeletal network. These findings suggest a logical, but unexpected, molecular mechanism underlying ion channel and transporter remodelling following MI.
The identification of nearly a dozen ion channel genes involved in the genesis of human atrial and ventricular arrhythmias has been critical for the diagnosis and treatment of fatal cardiovascular diseases. In contrast, very little is known about the genetic and molecular mechanisms underlying human sinus node dysfunction (SND). Here, we report a genetic and molecular mechanism for human SND. We mapped two families with highly penetrant and severe SND to the human ANK2 (ankyrin-B/AnkB) locus. Mice heterozygous for AnkB phenocopy human SND displayed severe bradycardia and rate variability. AnkB is essential for normal membrane organization of sinoatrial node cell channels and transporters, and AnkB is required for physiological cardiac pacing. Finally, dysfunction in AnkB-based trafficking pathways causes abnormal sinoatrial node (SAN) electrical activity and SND. Together, our findings associate abnormal channel targeting with human SND and highlight the critical role of local membrane organization for sinoatrial node excitability.
• calcium
• trafficking
• arrhythmia
• cytoskeleton
Aplysia S-type K+ channels of sensory neurons play a dominant role in presynaptic facilitation and behavioural sensitization. They are closed by serotonin via cAMP-dependent phosphorylation, whereas they are opened by arachidonic acid, volatile general anaesthetics and mechanical stimulation. We have identified a cloned mammalian two P domain K+ channel sharing the properties of the S channel. In addition, the recombinant channel is opened by lipid bilayer amphipathic crenators, while it is closed by cup-formers. The cytoplasmic C-terminus contains a charged region critical for chemical and mechanical activation, as well as a phosphorylation site required for cAMP inhibition.
The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca2+ channel α1C subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina,
T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901–17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties
in the genomic sequence of the N-terminal region of α1Cleave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate
the presence of α1C protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody.
Using deletion mutants of α1C expressed inXenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the
first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect
on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathioneS-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct
phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation
of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.
Recent evidence suggests that K(+) channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myocytes, and activation of the cAMP second messenger cascade has been shown to modulate this transient current. We determined if Kv4.2 alpha-subunits were directly phosphorylated by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that both of these fusion proteins were substrates for PKA in vitro. By using phosphopeptide mapping and amino acid sequencing, we identified PKA phosphorylation sites on the amino- and carboxyl-terminal fusion proteins corresponding to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate PKA site phosphorylation in situ, phospho-selective antisera for each of the sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed modulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal area CA1. These results provide insight into likely sites of regulation of Kv4.2 by PKA.
Neuroepithelial bodies act as airway oxygen sensors. The lung carcinoma line H146 is an established model for neuroepithelial body cells. Although O(2) sensing in both cells is via NADPH oxidase H(2)O(2)/free radical production and acute hypoxia promotes K(+) channel closure and cell depolarization, the identity of the K(+) channel is still controversial. However, recent data point toward the involvement of a member of the tandem P domain family of K(+) channels. Reverse transcription-polymerase chain reaction screening indicates that all known channels other than hTWIK1 and hTRAAK are expressed in H146 cells. Our detailed pharmacological characterization of the O(2)-sensitive K(+) current described herein is compatible with the involvement of hTASK1 or hTASK3 (pH dependence, tetraethylammonium and dithiothreitol insensitivity, blockade by arachidonic acid, and halothane activation). Furthermore, we have used antisense oligodeoxynucleotides directed against hTASK1 and hTASK3 to suppress almost completely the hTASK1 protein and show that these cells no longer respond to acute hypoxia; this behavior was not mirrored in liposome-only or missense-treated cells. Finally, we have used Zn(2+) treatment as a maneuver able to discriminate between these two homologues of hTASK and show that the most likely candidate channel for O(2) sensing in these cells is hTASK3.
The autosomal recessive mouse mutation quivering (qv), which arose spontaneously in 1953, produces progressive ataxia with hind limb paralysis, deafness and tremor. Six additional spontaneous alleles, qvJ, qv2J, qv3J, qv4J, qvlnd and qvlnd2J, have been identified. Ear twitch responses (Preyer's reflex) to sound are absent in homozygous qv/qv mice, although cochlear morphology seems normal and cochlear potentials recorded at the round window are no different from those of control mice. However, responses from brainstem auditory nuclei show abnormal transmission of auditory information, indicating that, in contrast to the many known mutations causing deafness originating in the cochlea, deafness in qv is central in origin. Here we report that quivering mice carry loss-of-function mutations in the mouse beta-spectrin 4 gene (Spnb4) that cause alterations in ion channel localization in myelinated nerves; this provides a rationale for the auditory and motor neuropathies of these mice.
beta-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a null mutation in the betaIV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. betaIV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In betaIV-spectrin-null neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G-null neurons, betaIV-spectrin is not localized to these sites. These results indicate that betaIV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.
Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.
TREK-1 is a two-pore-domain background potassium channel expressed throughout the central nervous system. It is opened by polyunsaturated fatty acids and lysophospholipids. It is inhibited by neurotransmitters that produce an increase in intracellular cAMP and by those that activate the Gq protein pathway. TREK-1 is also activated by volatile anesthetics and has been suggested to be an important target in the action of these drugs. Using mice with a disrupted TREK-1 gene, we now show that TREK-1 has an important role in neuroprotection against epilepsy and brain and spinal chord ischemia. Trek1-/- mice display an increased sensitivity to ischemia and epilepsy. Neuroprotection by polyunsaturated fatty acids, which is impressive in Trek1+/+ mice, disappears in Trek1-/- mice indicating a central role of TREK-1 in this process. Trek1-/- mice are also resistant to anesthesia by volatile anesthetics. TREK-1 emerges as a potential innovative target for developing new therapeutic agents for neurology and anesthesiology.
High densities of sodium channels at nodes of Ranvier permit action potential conduction and depend on betaIV spectrins, a family of scaffolding proteins linked to the cortical actin cytoskeleton. To investigate the molecular organization of nodes, we analyzed qv(3J)"quivering" mice, whose betaIV spectrins have a truncated proline-rich "specific" domain (SD) and lack the pleckstrin homology (PH) domain. Central nodes of qv(3J) mice, which lack betaIV spectrins, are significantly broader and have prominent vesicle-filled nodal membrane protrusions, whereas axon shape and neurofilament density are dramatically altered. PNS qv(3J) nodes, some with detectable betaIV spectrins, are less affected. In contrast, a larger truncation of betaIV spectrins in qv(4J) mice, deleting the SD, PH, and ankyrinG binding domains, causes betaIV spectrins to be undetectable and causes dramatic changes, even in peripheral nodes. These results show that quivering mutations disrupt betaIV spectrin retention and stability at nodes and that distinct protein domains regulate nodal structural integrity and molecular organization.
We identify a human mutation (E1053K) in the ankyrin-binding motif of Nav1.5 that is associated with Brugada syndrome, a fatal cardiac arrhythmia caused by altered function of Nav1.5. The E1053K mutation abolishes binding of Nav1.5 to ankyrin-G, and also prevents accumulation of Nav1.5 at cell surface sites in ventricular cardiomyocytes. Ankyrin-G and Nav1.5 are both localized at intercalated disc and T-tubule membranes in cardiomyocytes, and Nav1.5 coimmunoprecipitates with 190-kDa ankyrin-G from detergent-soluble lysates from rat heart. These data suggest that Nav1.5 associates with ankyrin-G and that ankyrin-G is required for Nav1.5 localization at excitable membranes in cardiomyocytes. Together with previous work in neurons, these results in cardiomyocytes suggest that ankyrin-G participates in a common pathway for localization of voltage-gated Nav channels at sites of function in multiple excitable cell types.
• arrhythmia
• SCN5A
• targeting
• trafficking
• sudden cardiac death
TREK-1 (KCNK2) is a K(2P) channel that is highly expressed in fetal neurons. This K(+) channel is opened by a variety of stimuli, including membrane stretch and cellular lipids. Here, we show that the expression of TREK-1 markedly alters the cytoskeletal network and induces the formation of actin- and ezrin-rich membrane protrusions. The genetic inactivation of TREK-1 significantly alters the growth cone morphology of cultured embryonic striatal neurons. Cytoskeleton remodelling is crucially dependent on the protein kinase A phosphorylation site S333 and the interactive proton sensor E306, but is independent of channel permeation. Conversely, the actin cytoskeleton tonically represses TREK-1 mechano-sensitivity. Thus, the dialogue between TREK-1 and the actin cytoskeleton might influence both synaptogenesis and neuronal electrogenesis.
Depression is a devastating illness with a lifetime prevalence of up to 20%. The neurotransmitter serotonin or 5-hydroxytryptamine (5-HT) is involved in the pathophysiology of depression and in the effects of antidepressant treatments. However, molecular alterations that underlie the pathology or treatment of depression are still poorly understood. The TREK-1 protein is a background K+ channel regulated by various neurotransmitters including 5-HT. In mice, the deletion of its gene (Kcnk2, also called TREK-1) led to animals with an increased efficacy of 5-HT neurotransmission and a resistance to depression in five different models and a substantially reduced elevation of corticosterone levels under stress. TREK-1-deficient (Kcnk2-/-) mice showed behavior similar to that of naive animals treated with classical antidepressants such as fluoxetine. Our results indicate that alterations in the functioning, regulation or both of the TREK-1 channel may alter mood, and that this particular K+ channel may be a potential target for new antidepressants.
Na/Ca exchanger activity is important for calcium extrusion from the cardiomyocyte cytosol during repolarization. Animal models exhibiting altered Na/Ca exchanger expression display abnormal cardiac phenotypes. In humans, elevated Na/Ca exchanger expression/activity is linked with pathophysiological conditions including arrhythmia and heart failure. Whereas the molecular mechanisms underlying Na/Ca exchanger biophysical properties are widely studied and generally well characterized, the cellular pathways and molecular partners underlying the specialized membrane localization of Na/Ca exchanger in cardiac tissue are essentially unknown. In this report, we present the first direct evidence for a protein pathway required for Na/Ca exchanger localization and stability in primary cardiomyocytes. We define the minimal structural requirements on ankyrin-B for direct Na/Ca exchanger interactions. Moreover, using ankyrin-B mutants that lack Na/Ca exchanger binding activity, and primary cardiomyocytes with reduced ankyrin-B expression, we demonstrate that direct interaction with the membrane adaptor ankyrin-B is required for the localization and post-translational stability of Na/Ca exchanger 1 in neonatal mouse cardiomyocytes. These results raise exciting new questions regarding potentially dynamic roles for ankyrin proteins in the biogenesis and maintenance of specialized membrane domains in excitable cells.
At axon initial segments and nodes of Ranvier in neurons, the spectrin membrane skeleton plays roles in physically stabilizing the plasma membrane integrity and in clustering voltage-gated sodium channels for proper conduction of the action potential. betaIV-Spectrin, an essential component of the membrane skeleton at these sites, has an N-terminal-truncated isoform, Sigma6, which is expressed at much higher levels than the full-length isoform Sigma1. To investigate the role of betaIV-spectrin Sigma6, we generated Sigma1-deficient mice with a normal level of Sigma6 expression (Sigma1(-/-) mice), and compared their phenotypes with those of previously generated mice lacking both Sigma1 and Sigma6(Sigma1Sigma6(-/-) mice). The gross neurological defects observed in Sigma1Sigma6(-/-) mice, such as hindleg contraction, were apparently ameliorated in Sigma1(-/-) mice. At cellular levels, Sigma1Sigma6(-/-) and Sigma1(-/-) neurons similarly exhibited waving and swelling of the plasma membrane at axon initial segments and nodes of Ranvier. By contrast, the levels of ankyrin G and voltage-gated sodium channels at these sites, which are significantly reduced in Sigma1Sigma6(-/-) mice, were substantially recovered in Sigma1(-/-) mice. We conclude that the truncated betaIV-spectrin isoform Sigma6 plays a specific role in clustering voltage-gated sodium channels, whereas it is dispensable for membrane stabilization at axon initial segments and nodes of Ranvier.
High densities of ion channels at axon initial segments (AISs) and nodes of Ranvier are required for initiation, propagation, and modulation of action potentials in axons. The organization of these membrane domains depends on a specialized cytoskeleton consisting of two submembranous cytoskeletal and scaffolding proteins, ankyrinG (ankG) and betaIV spectrin. However, it is not known which of these proteins is the principal organizer, or if the mechanisms governing formation of the cytoskeleton at the AIS also apply to nodes. We identify a distinct protein domain in betaIV spectrin required for its localization to the AIS, and show that this domain mediates betaIV spectrin's interaction with ankG. Dominant-negative ankG disrupts betaIV spectrin localization, but does not alter endogenous ankG or Na(+) channel clustering at the AIS. Finally, using adenovirus for transgene delivery into myelinated neurons, we demonstrate that betaIV spectrin recruitment to nodes of Ranvier also depends on binding to ankG.
The TWIK related K+ channel TREK1 is an important member of the class of two-pore-domain K+ channels. It is a background K+ channel and is regulated by hormones, neurotransmitters, intracellular pH and mechanical stretch. This work shows that TREK1 is present both in mesenteric resistance arteries and in skin microvessels. It is particularly well expressed in endothelial cells. Deletion of TREK1 in mice leads to an important alteration in vasodilation of mesenteric arteries induced by acetylcholine and bradykinin. Iontophoretic delivery of acetylcholine and bradykinin in the skin of TREK1+/+ and TREK1-/- mice also shows the important role of TREK1 in cutaneous endothelium-dependent vasodilation. The vasodilator response to local pressure application is also markedly decreased in TREK1-/- mice, mimicking the decreased response to pressure observed in diabetes. Deletion of TREK1 is associated with a marked alteration in the efficacy of the G-protein-coupled receptor-associated cascade producing NO that leads to major endothelial dysfunction.
The National Heart, Lung, and Blood Institute and Office of Rare Diseases at the National Institutes of Health organized a workshop (September 14 to 15, 2006, in Bethesda, Md) to advise on new research directions needed for improved identification and treatment of rare inherited arrhythmias. These included the following: (1) Na+ channelopathies; (2) arrhythmias due to K+ channel mutations; and (3) arrhythmias due to other inherited arrhythmogenic mechanisms. Another major goal was to provide recommendations to support, enable, or facilitate research to improve future diagnosis and management of inherited arrhythmias. Classifications of electric heart diseases have proved to be exceedingly complex and in many respects contradictory. A new contemporary and rigorous classification of arrhythmogenic cardiomyopathies is proposed. This consensus report provides an important framework and overview to this increasingly heterogeneous group of primary cardiac membrane channel diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular biology and novel therapeutic approaches in cardiology, as well as the introduction of many recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy in consensus with a recent American Heart Association Scientific Statement.
Voltage-gated Na(v) channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na(v)1.5 is the predominant Na(v) channel, and Na(v)1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na(v)1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na(v) channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na(v)1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na(v)1.5 expression, abnormal Na(v)1.5 membrane targeting, and reduced Na(+) channel current density. We define the structural requirements on ankyrin-G for Na(v)1.5 interactions and demonstrate that loss of Na(v)1.5 targeting is caused by the loss of direct Na(v)1.5-ankyrin-G interaction. These data are the first report of a cellular pathway required for Na(v) channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na(v) channel organization in multiple excitable tissues.
Transgenic mouse models provided a powerful tool to evaluate the physiological significance of altered quantities or characteristics of specific gene products, such as cardiac ion channels. We have developed a system to record and analyze changes in the electrocardiogram in the mouse using an implantable telemetry system. The R-R and Q-T intervals were measured on individual beats and on signalaveraged complexes derived from 1, 2, or 4 s of contiguous data each hour during a 24-h period in three male and three female FVB mice. Duration of averaging had minimal effect on the measured Q-T. The Q-T interval was shown to be related to the square root of the R-R interval, and an appropriate formula for a rate-corrected Q-T interval (Q-Tc) was derived. Ketamine anesthesia was shown to markedly increase duration and variability in R-R, Q-T, and Q-Tc intervals. In conscious animals, variability in Q-T was low across animals and over time, suggesting that this should be a sensitive model for detection of changes in the Q-T interval in transgenic mice with ion channel defects.
Two-pore-domain K(+) (K(2P)) channel subunits are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem and function as either homo- or heterodimeric channels. This structural motif is associated with unusual gating properties, including background channel activity and sensitivity to membrane stretch. Moreover, K(2P) channels are modulated by a variety of cellular lipids and pharmacological agents, including polyunsaturated fatty acids and volatile general anaesthetics. Recent in vivo studies have demonstrated that TREK1, the most thoroughly studied K(2P) channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.
Purpose
Aberrant expression of potassium (K+) channels contributes to cancer cell proliferation and apoptosis, and K+ channel blockers can inhibit cell proliferation. TREK-1 and -2 belong to the two-pore domain (K2P) superfamily. We report TREK-1 and -2 expression in ovarian cancer and normal ovaries, and the effects of TREK-1 modulators on cell proliferation and apoptosis.
Methods
The cellular localisation of TREK-1 and -2 was investigated by immunofluorescence in SKOV-3 and OVCAR-3 cell lines and in cultured ovarian surface epithelium and cancer. Channel expression in normal ovaries and cancer was quantified by western blotting. Immunohistochemical analysis demonstrated the association between channel expression and disease prognosis, stage, and grade. TREK-1 modulation of cell proliferation in the cell lines was investigated with the MTS-assay and the effect on apoptosis determined using flow cytometry.
Results
Expression was identified in both cell lines, ovarian cancer (n = 22) and normal ovaries (n = 6). IHC demonstrated positive staining for TREK-1 and -2 in 95.7 % of tumours (n = 69) and 100 % of normal ovaries (n = 9). A reduction in cell proliferation (P
Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na(+) channel (Na(v)1.5) by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated.
We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na(v)1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na(v)1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Na(v)1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium.
We identify the mechanism for 2 human arrhythmia variants that affect Na(v)1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Na(v)1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Na(v)1.5 in congenital and acquired cardiac disease.
Transgenic mouse models provided a powerful tool to evaluate the physiological significance of altered quantities or characteristics of specific gene products, such as cardiac ion channels. We have developed a system to record and analyze changes in the electrocardiogram in the mouse using an implantable telemetry system. The R-R and Q-T intervals were measured on individual beats and on signal-averaged complexes derived from 1, 2, or 4 s of contiguous data each hour during a 24-h period in three male and three female FVB mice. Duration of averaging had minimal effect on the measured Q-T. The Q-T interval was shown to be related to the square root of the R-R interval, and an appropriate formula for a rate-corrected Q-T interval (Q-Tc) was derived. Ketamine anesthesia was shown to markedly increase duration and variability in R-R, Q-T, and Q-Tc intervals. In conscious animals, variability in Q-T was low across animals and over time, suggesting that this should be a sensitive model for detection of changes in the Q-T interval in transgenic mice with ion channel defects.
Potassium (K(+) ) channels are important in cardiovascular disease both as drug targets and as a cause of underlying pathology. Voltage-dependent K(+) (K(V) ) channels are inhibited by the class III antiarrhythmic agents. Certain vasodilators work by opening K(+) channels in vascular smooth muscle cells (VSMCs), and K(+) channel activation may also be a route to improving endothelial function. The two-pore domain K(+) (K(2P) ) channels form a group of 15 known channels with an expanding list of functions in the cardiovascular system. One of these K(2P) channels, TREK-1, is the focus of this review. TREK-1 channel activity is tightly regulated by intracellular and extracellular pH, membrane stretch, polyunsaturated fatty acids (PUFAs), temperature, and receptor-coupled second messenger systems. TREK-1 channels are also activated by volatile anesthetics and some neuroprotectant agents, and they are inhibited by selective serotonin reuptake inhibitors (SSRIs) as well as amide local anesthetics. Some of the clinical cardiovascular effects and side effects of these drugs may be through their actions on TREK-1 channels. It has recently been suggested that TREK-1 channels have a role in mechano-electrical coupling in the heart. They also seem important in the vascular responses to PUFAs, and this may underlie some of the beneficial cardiovascular effects of the essential dietary fatty acids. Development of selective TREK-1 openers and inhibitors may provide promising routes for intervention in cardiovascular diseases.
By functional coexpression screening of a rat cDNA library in Xenopus oocytes, we have cloned a protein (KCRF: K Channel Regulatory Factor) that reduces currents of several K(+) channels: G protein-activated GIRK1/4 (K(ir)3.1/K(ir)3.4), inward rectifier IRK1 (K(ir)2.1), and voltage-dependent K(V)1.1/K(V)beta1.1. KCRF did not modulate two other K(+) channels: ROMK1 (K(ir)1.1) and GIRK1/2 (K(ir)3.1/K(ir)3.2) and the voltage-dependent L-type Ca(2+) channels. Western blot analysis showed that KCRF is ubiquitous in rat tissues. Biochemical and electrophysiological experiments revealed that coexpression of KCRF causes a decrease in the level of expression of IRK1 and K(V)1.1/K(V)beta1.1 proteins in the oocytes.
The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.
Many members of the two-pore-domain potassium (K(+)) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether I(KAA), a background K(+) current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that I(KAA), like TREK-1, was outwardly rectifying in physiological K(+) conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, I(KAA) was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, I(KAA) was inhibited by chlorophenylthio-cAMP (500 micromol/L) and also by stimulation of beta-adrenergic receptors with isoproterenol (1 micromol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by beta-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.
In the mammalian myocardium, potassium (K(+)) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K(+) channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K(+) channel pore forming (alpha) and accessory (beta) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K(+) channel subunits and functional cardiac K(+) channels. To date, more than 20 mouse models with altered K(+) channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K(+) channel subunit expression has revealed the role of specific K(+) channel subunit subfamilies or individual K(+) channel subunit genes in the generation of myocardial K(+) channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K(+) channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.
Large (111 +/- 3.0 pS) K+ channels were recorded in membrane patches from adult rat ventricular myocytes using patch-clamp techniques. The channels were not blocked by 4-AP (5 mM), intracellular TEA (5 mM) or glybenclamide (100 mM). Applying stretch to the membrane (as pipette suction) increased channel open probability (Po) in both cell-attached and isolated patches (typically, Po approximately equals 0.005 with no pressure; approximately equals 0.328 with 90 cm H2O: Vm = 40 mV, pHi = 7.2). The channels were activated by a decrease in intracellular pH; decreasing pHi to 5.5 from 7.2 increased Po to 0.16 from approx. 0.005 (no suction, Vm held at 40 mV). These properties are consistent with those demonstrated for TREK-1, a member of the recently cloned tandem pore family. We confirmed, using RT-PCR, that TREK-1 is expressed in rat ventricle, suggesting that the channel being recorded is indeed TREK-1. However, we show also that the channels are activated by millimolar concentrations of intracellular ATP. At a pH of 6 with no ATP at the intracellular membrane face, Po was 0.048 +/-0.023, whereas Po increased to 0.22 +/- 0.1 with 1 mM ATP, and to 0.348 +/- 0.13 with 3 mM (n = 5; no membrane stretch applied). The rapid time course of the response and the fact that we see the effect in isolated patches appear to preclude phosphorylation. We conclude that intracellular ATP directly activates TREK-like channels, a property not previously described.
Although the canine atrium has proven useful in several experimental models of atrial fibrillation and for studying the effects of rapid atrial pacing on atrial electrical remodeling, it may not fully represent the human condition because of reported differences in functional ionic currents and ion channel subunit expression. In this study, we reassessed the molecular components underlying one current, the ultrarapid delayed rectifier current in canine atrium [IKur(d)], by evaluating the mRNA, protein, immunofluorescence, and currents of the candidate channels. Using reverse transcriptase-polymerase chain reaction, we found that Kv1.5 mRNA was expressed in canine atrium whereas message for Kv3.1 was not detected. Western analysis on cytosolic and membrane fractions of canine tissues, using selective antibodies, showed that Kv3.1 was only detectable in the brain preparations, whereas Kv1.5 was expressed at high levels in both atrial and ventricular membrane fractions. Confocal imaging performed on isolated canine atrial myocytes clearly demonstrated the presence of Kv1.5 immunostaining, whereas that of Kv3.1 was equivocal. Voltage- and current-clamp studies showed that 0.5 mmol/L tetraethylammonium had variable effects on sustained K+ currents, whereas a compound with demonstrated selectivity for hKv1.5 versus Kv3.1, hERG or the sodium channel, fully suppressed canine atrial IKur tail currents and depressed sustained outward K+ current. This agent also increased action potential plateau potentials and action potential duration at 20% and 50% repolarization. These results suggest that in canine atria, as in other species including human, Kv1.5 protein is highly expressed and contributes to IKur.
Previous studies have documented the expression of four kinetically distinct voltage-gated K(+) (Kv) currents, I(to,f), I(to,s), I(K,slow) and I(ss), in mouse ventricular myocytes and demonstrated that I(to,f) and I(to,s) are differentially expressed in the left ventricular apex and the interventricular septum. The experiments here were undertaken to test the hypothesis that there are further regional differences in the expression of Kv currents or the Kv subunits (Kv4.2, Kv4.3, KChIP2, Kv1.5, Kv2.1) encoding these currents in adult male and female (C57BL6) mouse ventricles. Whole-cell voltage-clamp recordings revealed that mean (+/-s.e.m.) peak outward K(+) current and I(to,f) densities are significantly (P < 0.001) higher in cells isolated from the right (RV) than the left (LV) ventricles. Within the LV, peak outward K(+) current and I(to,f) densities are significantly (P < 0.05) higher in cells from the apex than the base. In addition, I(to,f) and I(K,slow) densities are lower in cells isolated from the endocardial (Endo) than the epicardial (Epi) surface of the LV wall. Importantly, similar to LV apex cells, I(to,s) is not detected in RV, LV base, LV Epi or LV Endo myocytes. No measurable differences in K(+) current densities or properties are evident in RV or LV cells from adult male and female mice, although I(to,f), I(to,s), I(K,slow) and I(ss) densities are significantly (P < 0.01) higher, and action potential durations at 50% (APD(50)) are significantly (P < 0.05) shorter in male septum cells. Western blot analysis revealed that the expression levels of Kv4.2, Kv4.3, KChIP2, Kv1.5 and Kv2.1 are similar in male and female ventricles. In addition, consistent with the similarities in repolarizing Kv current densities, no measurable differences in ECG parameters, including corrected QT (QT(c)) intervals, are detected in telemetric recordings from adult male and female (C57BL6) mice.
The biophysical properties and the regulation of the two-pore-domain potassium channel TREK-1 were studied in rat cardiomyocytes.
RT-PCR, immunohistochemistry and patch-clamp recording were performed in isolated rat ventricular cardiomyocytes. In some whole-cell-clamp experiments the myocytes were mechanically stretched using a glass stylus.
We found strong expression of a splice variant of TREK-1 in rat heart. Immunohistochemistry with antibodies against TREK-1 showed localization of the channel in longitudinal stripes at the external surface membrane of cardiomyocytes. When the cardiomyocytes were mechanically stretched, an outwardly rectifying K+ current component could be detected in whole-cell recordings. In single-channel recordings with symmetrical high K+ solution, two TREK-like channels with 'flickery-burst' kinetics were found: a 'large conductance' K+ channel (132+/-5 pS at positive potentials) and a novel 'low-conductance' channel (41+/-5 pS at positive potentials). The low-conductance channel could be activated by negative pressure in inside-out patches, positive pressure in outside-out patches, intracellular acidification and application of arachidonic acid. Its open probability was strongly increased by depolarization, due to decreased duration of gaps between bursts. The biophysical properties of the two cardiac TREK-like channels were similar to those of TREK-1 channels expressed in HEK293 cells, which both displayed low- and high-conductance modes.
Our results suggest that the two TREK-like channels found in rat cardiomyocytes may reflect two different operating modes of TREK-1. The novel low-conductance channels described here may represent the major operating mode of TREK-1. The current flowing through mechanogated TREK-1 channels may serve to counterbalance the inward current flowing through stretch-activated non-selective cation channels during the filling phase of the cardiac cycle and thus to prevent the occurrence of ventricular extrasystoles.
Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether beta-adrenergic receptor (beta-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca](i)) and SR Ca load ([Ca](SRT)) were assessed using fluo-4. beta-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca](i) decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2-related inhibitory peptide II or beta-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the beta-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca](i) decline rate. The results suggest that beta-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca](i).
Two-pore-domain K(+) (K(2P)) channel subunits are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem and function as either homo- or heterodimeric channels. This structural motif is associated with unusual gating properties, including background channel activity and sensitivity to membrane stretch. Moreover, K(2P) channels are modulated by a variety of cellular lipids and pharmacological agents, including polyunsaturated fatty acids and volatile general anaesthetics. Recent in vivo studies have demonstrated that TREK1, the most thoroughly studied K(2P) channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.
Rhythmic and effective cardiac contraction depends on appropriately timed generation and spread of cardiac electrical activity. The basic cellular unit of such activity is the action potential, which is shaped by specialized proteins (channels and transporters) that control the movement of ions across cardiac cell membranes in a highly regulated fashion. Cardiac disease modifies the operation of ion channels and transporters in a way that promotes the occurrence of cardiac rhythm disturbances, a process called "arrhythmogenic remodeling." Arrhythmogenic remodeling involves alterations in ion channel and transporter expression, regulation and association with important protein partners, and has important pathophysiological implications that contribute in major ways to cardiac morbidity and mortality. We review the changes in ion channel and transporter properties associated with three important clinical and experimental paradigms: congestive heart failure, myocardial infarction, and atrial fibrillation. We pay particular attention to K+, Na+, and Ca2+ channels; Ca2+ transporters; connexins; and hyperpolarization-activated nonselective cation channels and discuss the mechanisms through which changes in ion handling processes lead to cardiac arrhythmias. We highlight areas of future investigation, as well as important opportunities for improved therapeutic approaches that are being opened by an improved understanding of the mechanisms of arrhythmogenic remodeling.
Ischemic preconditioning delays the onset of electrical uncoupling and prevents loss of the primary ventricular gap junction protein connexin 43 (Cx43) from gap junctions during subsequent ischemia.
To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKCepsilon), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCepsilon (PKCepsilon-KO).
Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy. Changes in electrical coupling were monitored using the 4-electrode technique to measure whole-tissue resistivity.
The amount of Cx43 located in gap junctions, measured by confocal microscopy under basal conditions, was significantly greater in PKCepsilon-KO hearts compared with wild-type, but total Cx43 content measured by immunoblotting was not different. These unanticipated results indicate that PKCepsilon regulates subcellular distribution of Cx43 under normal conditions. Preconditioning prevented loss of Cx43 from gap junctions during ischemia in wild-type but not PKCepsilon-KO hearts. Specific activation of PKCepsilon, but not PKCdelta, also prevented ischemia-induced loss of Cx43 from gap junctions. Preconditioning delayed the onset of uncoupling in wild-type but hastened uncoupling in PKCepsilon-KO hearts. Cx43 phosphorylation at the PKC site Ser368 increased 5-fold after ischemia in wild-type hearts, and surprisingly, by nearly 10-fold in PKCepsilon-KO hearts. Preconditioning prevented phosphorylation of Cx43 in gap junction plaques at Ser368 in wild-type but not PKCepsilon-KO hearts.
Taken together, these results indicate that PKCepsilon plays a critical role in preconditioning to preserve Cx43 signal in gap junctions and delay electrical uncoupling during ischemia.
Two-pore domain (K(2P)) channels emerged about a decade ago and since then have been an expanding area of interest. This is because their biophysical and pharmacological properties make them good candidates to support background potassium currents and membrane potential in many cell types. There is clear evidence for TREK-1 and TASK-1 in the heart and these channels are likely to regulate cardiac action potential duration through their regulation by stretch, polyunsaturated fatty acids, pH, and neurotransmitters. TREK-1 may also have a critical role in mediating the vasodilator response of resistance arteries to polyunsaturated fatty acids, thus contributing to their protective effect on the cardiovascular system. TASK-1, on the other hand, is a strong candidate for a role in hypoxic vasoconstriction of pulmonary arteries. Many other members of the K(2P) channel family have been identified in the cardiovascular system, although their functional roles are still to be demonstrated. This review provides an up to date summary of what is known about the involvement of members of the K(2P) channel family in cells of the heart and arterial circulation. Our knowledge of their roles will improve with the rapidly increasing interest in them and as new selective pharmacological tools emerge. As their physiological roles emerge, the K(2P) family of potassium channels may offer promising therapeutic solutions to target cardiovascular diseases.