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Arrhythmia modeling with mathematical Purkinje cells. Membrane potential during 300-ms basic cycle length (BCL) stimulations followed by a pause is shown in (A) control conditions, (B) with I K1 downregulation (−50%), (C) I NaL enhancement (2-fold), or (D) with concomitant I K1 reduction and I NaL increase (fourth row). E, Triggered activity in the latter condition cannot be prevented by I f inhibition.
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Background:
In human cardiac ventricle, IK1 is mainly comprised Kir2.1, but Kir2.2 and Kir2.3 heterotetramers occur and modulate IK1. Long-QT syndrome-9-associated CAV3 mutations cause decreased Kir2.1 current density, but Kir2.x heterotetramers have not been studied. Here, we determine the effect of long-QT syndrome-9-CAV3 mutation F97C on Kir2.x...
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Context 1
... dual effect of calcium loading and unstable rest- ing membrane potential increases the likelihood of calcium overload-mediated diastolic calcium release and decreases the threshold for DADs as shown in Figure 7. Notably, blocking I f does not prevent the occurrence of unstimulated beats. At faster pacing rates, under control conditions or with 50% I K1 reduc- tion alone, no triggered activity is observed, and with 50% I K1 reduction, there is slight resting membrane potential depolarization ( Figure 8A and 8B). When simulating I NaL upregulation (+100%) alone, DAD- mediated triggered activity is predicted causing a few unstimulated beats before returning to a stable rest- ing membrane potential ( Figure 8C). ...
Context 2
... faster pacing rates, under control conditions or with 50% I K1 reduc- tion alone, no triggered activity is observed, and with 50% I K1 reduction, there is slight resting membrane potential depolarization ( Figure 8A and 8B). When simulating I NaL upregulation (+100%) alone, DAD- mediated triggered activity is predicted causing a few unstimulated beats before returning to a stable rest- ing membrane potential ( Figure 8C). Notably, when concomitant I K1 downregulation and I NaL enhance- ment are combined, as is the hypothesized effect of F97C-Cav3 mutation, the synergistic interaction of increasing calcium load and reducing DAD threshold lead to AP oscillations, which are not prevented by I f inhibition ( Figure 8D and 8E). ...
Context 3
... simulating I NaL upregulation (+100%) alone, DAD- mediated triggered activity is predicted causing a few unstimulated beats before returning to a stable rest- ing membrane potential ( Figure 8C). Notably, when concomitant I K1 downregulation and I NaL enhance- ment are combined, as is the hypothesized effect of F97C-Cav3 mutation, the synergistic interaction of increasing calcium load and reducing DAD threshold lead to AP oscillations, which are not prevented by I f inhibition ( Figure 8D and 8E). ...
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The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cer...
Citations
... Moreover, both channels have been shown to co-immunoprecipitate in both the forward and reverse coimmunoprecipitation reactions (Ponce-Balbuena et al., 2018). Independent studies by Eckhardt and Jalife have demonstrated that K IR 2.1 and Na V 1.5, colocalize in ventricular myocytes in humans, rats, and mice (Milstein et al., 2012;Vaidyanathan et al., 2018). ...
In cardiac cells, the expression of the cardiac voltage-gated Na⁺ channel (NaV1.5) is reciprocally regulated with the inward rectifying K⁺ channel (KIR2.1). These channels can form macromolecular complexes that pre-assemble early during forward trafficking (transport to the cell membrane). In this study, we present in silico 3D models of NaV1.5-KIR2.1, generated by rigid-body protein-protein docking programs and deep learning-based AlphaFold-Multimer software. Modeling revealed that the two channels could physically interact with each other along the entire transmembrane region. Structural mapping of disease-associated mutations revealed a hotspot at this interface with several trafficking-deficient variants in close proximity. Thus, examining the role of disease-causing variants is important not only in isolated channels but also in the context of macromolecular complexes. These findings may contribute to a better understanding of the life-threatening cardiovascular diseases underlying KIR2.1 and NaV1.5 malfunctions.
... Consistent with previous studies in multiple mammalian species, we observed NaV1.5 6, 44-47 , Kir2.1 6,[48][49][50][51][52][53] , and NKA (α1 subunit) 11, 54-56 localizing to ID, lateral sarcolemma and transverse tubules using confocal microscopy, with NaV1.5, in particular, showing marked enrichment at the ID. However, these previous reports were largely qualitative, precluding direct comparisons with our quantitative measurements. ...
During each heartbeat, the propagation of action potentials through the heart coordinates the contraction of billions of individual cardiomyocytes and is thus, a critical life process. Unsurprisingly, intercalated discs, which are cell-cell contact sites specialized to provide electrical and mechanical coupling between adjacent cardiomyocytes, have been the focus of much investigation. Slowed or disrupted propagation leads to potentially life-threatening arrhythmias in a wide range of pathologies, where intercalated disc remodeling is a common finding. Hence, the importance and urgency of understanding intercalated disc structure and its influence on action potential propagation. Surprisingly, however, conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, owing to lack of quantitative structural data at subcellular through nano scales. In order to address this critical gap in knowledge, we sought to quantify intercalated disc structure at these finer spatial scales in the healthy adult mouse heart and relate them to function in a chamber-specific manner as a precursor to understanding the impacts of pathological intercalated disc remodeling. Using super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization. By incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by inter-chamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction. These data provide the first stepping stone to elucidating chamber-specific impacts of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.
... While these findings indicate that there is a fundamental difference between dystrophin deficiency and loss of the SIV domain in Na v 1.5 required for dystrophin binding, the underlying mechanisms remain to be elucidated. In contrast to the loss-of-function related to DMD, mutations in LM-specific Na V 1.5 interacting proteins α-1-syntrophin (SNTA1) and caveolin-3 (CAV3) have been associated with a long QT syndrome phenotype and a gain-of-function of Na V 1.5 consisting of an increased I Na,L via neuronal nitric oxide synthase-mediated nitrosylation of Na V 1.5 (Vaidyanathan et al., 2018;Vatta et al., 2006;Wu, Ai et al., 2008). While the impact of these mutations on distinct subcellular Na V 1.5 (dys)function has not been assessed, it is tempting to speculate that the observed gain-of-function is mediated specifically by LM-based Na V 1.5 channels, although co-existing effects on other ion channels likely also play a role. ...
... While the impact of these mutations on distinct subcellular Na V 1.5 (dys)function has not been assessed, it is tempting to speculate that the observed gain-of-function is mediated specifically by LM-based Na V 1.5 channels, although co-existing effects on other ion channels likely also play a role. Indeed, caveolin-3 also associates with potassium inward rectifier channels (K ir 2.x), and mutations in CAV3 may decrease inward rectifying K + current (I K1 ) and thereby affect membrane potential, excitability and repolarisation (Vaidyanathan et al., 2018). Interestingly, caveolin-3 also interacts with the dystrophin-associated glycoprotein complex and dystrophin, and increased caveolin-3 protein expression has been observed in tissue from DMD patients and mdx mice (Pradhan & Prószyński, 2020); whether this enhanced expression contributes to changes in Na V 1.5 and/or I Na is as yet unknown. ...
In cardiomyocytes, the rapid depolarisation of the membrane potential is mediated by the α‐subunit of the cardiac voltage‐gated Na⁺ channel (NaV1.5), encoded by the gene SCN5A. This ion channel allows positively charged Na⁺ ions to enter the cardiomyocyte, resulting in the fast upstroke of the action potential and is therefore crucial for cardiac excitability and electrical propagation. This essential role is underscored by the fact that dysfunctional NaV1.5 is associated with high risk for arrhythmias and sudden cardiac death. However, development of therapeutic interventions regulating NaV1.5 has been limited due to the complexity of NaV1.5 structure and function and its diverse roles within the cardiomyocyte. In particular, research from the last decade has provided us with increased knowledge on the subcellular distribution of NaV1.5 as well as the proteins which it interacts with in distinct cardiomyocyte microdomains. We here review these insights, detailing the potential role of NaV1.5 within subcellular domains as well as its dysfunction in the setting of arrhythmia disorders. We furthermore provide an overview of current knowledge on the pathways involved in (microdomain‐specific) trafficking of NaV1.5, and their potential as novel targets. Unravelling the complexity of NaV1.5 (dys)function may ultimately facilitate the development of therapeutic strategies aimed at preventing lethal arrhythmias. This is not only of importance for pathophysiological conditions where sodium current is specifically decreased within certain subcellular regions, such as in arrhythmogenic cardiomyopathy and Duchenne muscular dystrophy, but also for other acquired and inherited disorders associated with NaV1.5. image
... Modeling loss of I K1 combined with gain of late I Na in a purkinje cell model resulted in DADs and sustained arrhythmia. 23 In contrast, the same conditions in a ventricular myocyte model resulted in EAD induction without sustained arrhythmia. Thus, tissue heterogeneity may be part of the missing link to arrhythmia generation with I K1 loss. ...
... As expected, mutations are highly deleterious within the Flag-tag used for surface labeling and known trafficking signals ( Figure 2B). Substituting aromatic residues with charged residues strongly decreases surface expression within the 81 WRWMLLLLF 88 motif that mediates interaction with Caveolin-3 (Vaidyanathan et al., 2018). Glutamates within the di-acidic ER export signal (Ma et al., 2001), 382 FCYENE 387 , are quite sensitive to mutations with the second glutamate being far more sensitive. ...
... In this region, beneficial mutations have no clear preferences beyond non-aromatics. While no known trafficking signal matches, aromatic residues are often found in binding interfaces (Vaidyanathan et al., 2018;Williams and Fukuda, 1990). Perhaps mutations alter trafficking patterns by disrupting protein interactions (e.g., reduce ER retention, reduce forwarding to lysosomal recycling). ...
A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutations into the Inward Rectifier K ⁺ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provide high-resolution information on a cotranslationally-folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 surface trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of 'variants of unknown significance' and disease mechanisms of known pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.
... Kir2.x family have a unique intracellular pattern of distribution in association with specific caveolin-3 domains, which critically depends on interaction with Kir2.x-caveolin-3 binding motifs [36]. An early study demonstrated that CAV-3 gene mutation decreased the surface expression of Kir2.1 and Kir2.2 [36,37]. ...
... Kir2.x family have a unique intracellular pattern of distribution in association with specific caveolin-3 domains, which critically depends on interaction with Kir2.x-caveolin-3 binding motifs [36]. An early study demonstrated that CAV-3 gene mutation decreased the surface expression of Kir2.1 and Kir2.2 [36,37]. Its mechanism may be closely related to the disruption in normal membrane trafficking of caveolin-3, regulating cell surface expression of Kir2.x channels, then causing decreased Kir2.x current density. ...
Caveolin-3 is a muscle-specific protein on the membrane of myocytes correlated with a variety of cardiovascular diseases. It is now clear that the caveolin-3 plays a critical role in the cardiovascular system and a significant role in cardiac protective signaling. Mutations in the gene encoding caveolin-3 cause a broad spectrum of clinical phenotypes, ranging from persistent elevations in the serum levels of creatine kinase in asymptomatic humans to cardiomyopathy. The influence of Caveolin-3(CAV-3) mutations on current density parallels the effect on channel trafficking. For example, mutations in the CAV-3 gene promote ventricular arrhythmogenesis in long QT syndrome 9 by a combined decrease in the loss of the inward rectifier current (IK1) and gain of the late sodium current (INa-L). The functional significance of the caveolin-3 has proved that caveolin-3 overexpression or knockdown contributes to the occurrence and development of arrhythmias. Caveolin-3 overexpression could lead to reduced diastolic spontaneous Ca2+ waves, thus leading to the abnormal L-Type calcium channel current-induced ventricular arrhythmias. Moreover, CAV-3 knockdown resulted in a shift to more negative values in the hyperpolarization-activated cyclic nucleotide channel 4 current (IHCN4) activation curve and a significant decrease in IHCN4 whole-cell current density. Recent evidence indicates that caveolin-3 plays a significant role in adipose tissue and is related to obesity development. The role of caveolin-3 in glucose homeostasis has attracted increasing attention. This review highlights the underlining mechanisms of caveolin-3 in arrhythmia. Progress in this field may contribute to novel therapeutic approaches for patients prone to developing arrhythmia.
... In addition to these considerations, cholesterol/flotillin-rich membranes are associated with caveolins, which are TM proteins that promote membrane curvature during caveolaemediated internalization. Kir2.1 interacts with caveolins Cav1, Cav2, and Cav3 (Ambrosini et al., 2014;Tikku et al., 2007;Han et al., 2014;Vaidyanathan et al., 2018;Ikezu et al., 1998). Caveolae, or little 'caves' within the PM, are associated with elevated cholesterol and control the endocytosis of many membrane proteins, including several K + channels. ...
... Caveolae, or little 'caves' within the PM, are associated with elevated cholesterol and control the endocytosis of many membrane proteins, including several K + channels. Although Cav1 is a negative regulator of Kir2.1 and Cav3 mutations can themselves cause long-QT syndrome, perhaps due to reduced Kir function, the role of caveolae in Kir2.x endocytosis remains largely unexplored (Han et al., 2014;Vaidyanathan et al., 2018). The binding site for Cav3 in Kir2.1 resides between amino acids 81-88 in the N-terminus of this protein, where a canonical ΩxΩxxxxΩ Cav3-binding motif is located (Ω is any aromatic amino acid; 81 WRWMLVIF 88 for Kir2.1 and shown as CBS in Figure 1A) (Vaidyanathan et al., 2018). ...
... Although Cav1 is a negative regulator of Kir2.1 and Cav3 mutations can themselves cause long-QT syndrome, perhaps due to reduced Kir function, the role of caveolae in Kir2.x endocytosis remains largely unexplored (Han et al., 2014;Vaidyanathan et al., 2018). The binding site for Cav3 in Kir2.1 resides between amino acids 81-88 in the N-terminus of this protein, where a canonical ΩxΩxxxxΩ Cav3-binding motif is located (Ω is any aromatic amino acid; 81 WRWMLVIF 88 for Kir2.1 and shown as CBS in Figure 1A) (Vaidyanathan et al., 2018). ...
Potassium (K+) homeostasis is tightly regulated for optimal cell and organismal health. Failure to control potassium balance results in disease, including cardiac arrythmias and developmental disorders. A family of inwardly rectifying potassium (Kir) channels helps cells maintain K+ levels. Encoded by KCNJ genes, Kir channels are comprised of a tetramer of Kir subunits, each of which contains two-transmembrane domains. The assembled Kir channel generates an ion selectivity filter for K+ at the monomer interface, which allows for K+ transit. Kir channels are found in many cell types and influence K+ homeostasis across the organism, impacting muscle, nerve and immune function. Kir2.1 is one of the best studied family members with well-defined roles in regulating heart rhythm, muscle contraction and bone development. Due to their expansive roles, it is not surprising that Kir mutations lead to disease, including cardiomyopathies, and neurological and metabolic disorders. Kir malfunction is linked to developmental defects, including underdeveloped skeletal systems and cerebellar abnormalities. Mutations in Kir2.1 cause the periodic paralysis, cardiac arrythmia, and developmental deficits associated with Andersen-Tawil Syndrome. Here we review the roles of Kir family member Kir2.1 in maintaining K+ balance with a specific focus on our understanding of Kir2.1 channel trafficking and emerging roles in development and disease. We provide a synopsis of the vital work focused on understanding the trafficking of Kir2.1 and its role in development.
... The K15N mutation is located in the N-terminus, which is highly conserved in the CAV3 protein and may impact protein configuration (9,26). Mutations in this region may impair the location and maturity of the CAV3 protein in the cell membrane (27,28). This may induce effects on the signaling pathways, which are associated with this protein (29). ...
Caveolin-3 (CAV3) is a muscle-specific protein present within the muscle cell membrane that affects signaling pathways, including the insulin signaling pathway. A previous assessment of patients with newly developed type 2 diabetes (T2DM) demonstrated that CAV3 gene mutations may lead to changes in protein secondary structure. A severe CAV3 P104L mutation has previously been indicated to influence the phosphorylation of skeletal muscle cells and result in impaired glucose metabolism. In the present study, the effect of CAV3 K15N gene transfection in C2C12 cells was assessed. Transfection with K15N reduced the expression of total CAV3 and AKT2 proteins in the cells, and the translocation of glucose transporter type 4 to the muscle cell membrane, which resulted in decreased glucose uptake and glycogen synthesis in myocytes. In conclusion, these results indicate that the CAV3 K15N mutation may cause insulin-stimulated impaired glucose metabolism in myocytes, which may contribute to the development of T2DM.
... Several discrete domains within the K IR 2.1 sequence have been associated with certain functions, like potassium selectivity [amino acid (aa) 144-146], Endoplasmic Reticulum (ER) export (aa 374-379; Ma et al., 2001;Stockklausner et al., 2001), Golgi export (aa 44-61 and 314-322; Hofherr et al., 2005;Ma et al., 2011), a PDZ binding domain (aa 425-427, Leonoudakis et al., 2004), a Caveolin3 binding motif (aa 81-88; Vaidyanathan et al., 2018). K IR 2.1 and K IR 2.2 crystal structure and homology modeling provided additional 3-dimensional information and showed a K IR 2.1 channel containing a transmembrane pore domain with a long intracellular pore extension formed by the so-called cytoplasmic pore domain (Pegan et al., 2005;Hansen et al., 2011;Lee et al., 2013). ...
KIR2.1 potassium channels, producing inward rectifier potassium current (I
K1
), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the "epestfind" webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I
K1
, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I
K1
channels.
... The shape and size of these caveolar structures depend on the isoform of caveolin because flask-shaped invaginating caveola are fourfold more densely packed with caveolin-1 than are flat caveola, and the density of caveolin-1 is sensitive to osmotic tensions [60]. Super-resolution microscopy also revealed signaling-mediated receptor rearrangements at caveolae, as shown by SIM for the purinergic receptor P2X7R in osteoblasts [61], by CLEM and STED for the Ca 2+ release channel ryanodine receptor at the sarcoplasmic reticulum in muscles [62,63], and by STED for the inward rectifier potassium channel Kir2.1 in human cardiac ventricles [64]. [46,47], atomic force microscopy (AFM) [47], or polarized total internal reflection fluorescence microscopy (pol-TIRF) [47] have been used to determine the curvature of endocytic membranes. ...
Since their invention about two decades ago, super-resolution microscopes have become a method of choice in cell biology. Owing to a spatial resolution below 50 nm, smaller than the size of most organelles, and an order of magnitude better than the diffraction limit of conventional light microscopes, super-resolution microscopy is a powerful technique for resolving intracellular trafficking. In this review we discuss discoveries in endocytosis and phagocytosis that have been made possible by super-resolution microscopy - from uptake at the plasma membrane, endocytic coat formation, and cytoskeletal rearrangements to endosomal maturation. The detailed visualization of the diverse molecular assemblies that mediate endocytic uptake will provide a better understanding of how cells ingest extracellular material.
