RESUMO
We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of â¼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an â¼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.
Assuntos
Epilepsia , Membrana Celular/metabolismo , Criança , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/metabolismo , Mutação , Potássio/metabolismo , Canais de Potássio/metabolismoRESUMO
We recently established a role for the stretch-activated two-pore-domain K+ (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-α-induced acute lung injury. We have now discovered the expression of large conductance, Ca2+-activated K+ (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model. As expected, LPS induced IL-6 and CCL-2 secretion from pulmonary endothelial and epithelial cells. BK activation with NS1619 decreased LPS-induced CCL-2 but not IL-6 secretion from endothelial cells and had no effect on epithelial cells, although fluorometric assays revealed that BK activation hyperpolarized the plasma membrane potential (Em) of both cell types. Interestingly, BK inhibition (Paxilline) did not alter cytokine secretion or the Em in either cell type. Furthermore, LPS treatment by itself did not affect the Em or intracellular Ca2+ concentrations. Therefore, we propose BK channel activation as a novel targeted approach to counteract LPS-induced CCL-2 secretion from endothelial cells. This protective effect appears to occur via Em hyperpolarization but independent of intracellular Ca2+ concentrations.
Assuntos
Células Epiteliais Alveolares/metabolismo , Quimiocina CCL2/metabolismo , Células Endoteliais/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Pulmão/metabolismo , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células HEK293 , Humanos , Hiperóxia/induzido quimicamente , Hiperóxia/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismoRESUMO
The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na(+) and Ca(2+) ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.
Assuntos
Citoplasma/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Cisteína/genética , Citoplasma/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Íons , Mesilatos/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Trocador de Sódio e Cálcio/química , XenopusRESUMO
This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) moved out, NCX is one of the main Na(+) influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na(+) ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na(+) and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na(+) homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field.
Assuntos
Potenciais de Ação , Arritmias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Congressos como Assunto , Humanos , Miócitos Cardíacos/fisiologiaRESUMO
Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.
Assuntos
Cálcio/metabolismo , Multimerização Proteica , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Animais , Sítios de Ligação/genética , Cálcio/farmacologia , Membrana Celular/metabolismo , Citoplasma/metabolismo , Cães , Feminino , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Conformação Proteica/efeitos dos fármacos , Trocador de Sódio e Cálcio/genética , Xenopus laevisRESUMO
Biological systems, particularly the brain, are frequently analyzed as networks, conveying mechanistic insights into their function and pathophysiology. This is the first study of a functional network of cardiac tissue. We use calcium imaging to obtain two functional networks in a subsidiary but essential pacemaker of the heart, the atrioventricular node (AVN). The AVN is a small cellular structure with dual functions: a) to delay the pacemaker signal passing from the sinoatrial node (SAN) to the ventricles, and b) to serve as a back-up pacemaker should the primary SAN pacemaker fail. Failure of the AVN can lead to syncope and death. We found that the shortest path lengths and clustering coefficients of the AVN are remarkably similar to those of the brain. The network is ``small-world," thus optimized for energy use vs transmission efficiency. We further study the network properties of AVN tissue with knock-out of the sodium-calcium exchange transporter. In this case, the average shortest path-lengths remained nearly unchanged showing network resilience, while the clustering coefficient was somewhat reduced, similar to schizophrenia in brain networks. When we removed the global action potential using principal component analysis (PCA) in wild-type model, the network lost its ``small-world" characteristics with less information-passing efficiency due to longer shortest path lengths but more robust signal propagation resulting from higher clustering. These two wild-type networks (with and without global action potential) may correspond to fast and slow conduction pathways. Laslty, a one-parameter non-linear preferential attachment model is a good fit to all three AVN networks.
RESUMO
The Na+-Ca2+ exchanger (NCX1) is the dominant Ca2+ extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na+ via a process known as Na+-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na+-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca2+ transients, and depressed cell shortening. The results demonstrate that NCX1 Na+-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility.
Assuntos
Potenciais de Ação , Cálcio , Miócitos Cardíacos , Trocador de Sódio e Cálcio , Sódio , Trocador de Sódio e Cálcio/metabolismo , Trocador de Sódio e Cálcio/genética , Animais , Miócitos Cardíacos/metabolismo , Masculino , Sódio/metabolismo , Camundongos , Cálcio/metabolismo , Contração Miocárdica/fisiologia , Contração Miocárdica/genética , Coração/fisiologia , Humanos , Mutação , Sistemas CRISPR-CasRESUMO
Sodium-calcium exchange (NCX) is the major calcium (Ca) efflux mechanism of ventricular cardiomyocytes. Consequently the exchanger plays a critical role in the regulation of cellular Ca content and hence contractility. Reductions in Ca efflux by the exchanger, such as those produced by elevated intracellular sodium (Na) in response to cardiac glycosides, raise sarcoplasmic reticulum (SR) Ca stores. The result is an increased Ca transient and cardiac contractility. Enhanced Ca efflux activity by the exchanger, for example during heart failure, may reduce diadic cleft Ca and excitation-contraction (EC) coupling gain. This aggravates the impaired contractility associated with SR Ca ATPase dysfunction and reduced SR Ca load in failing heart muscle. Recent data from our laboratories indicate that NCX can also impact the efficiency of EC coupling and contractility independent of SR Ca load through diadic cleft priming with Ca during the upstroke of the action potential. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".
Assuntos
Cálcio/metabolismo , Acoplamento Excitação-Contração , Contração Miocárdica , Sódio/metabolismo , Potenciais de Ação , Animais , Transporte Biológico , Estruturas da Membrana Celular/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
The plasma membrane Na(+)/Ca(2+) exchanger (NCX) plays a critical role in the maintenance of Ca(2+) homeostasis in a variety of tissues. NCX accomplishes this task by either lowering or increasing the intracellular Ca(2+) concentration, a process which depends on electrochemical gradients. During each cycle, three Na(+) are transported in the opposite direction to one Ca(2+), resulting in an electrogenic transport that can be measured as an ionic current.The residues involved in ion translocation are unknown. A residue thought to be important for Na(+) and/or Ca(2+) transport, Ser(110), was replaced with a cysteine, and the properties of the resulting exchanger mutant were analyzed using the giant patch technique. Data indicate that this residue, located in transmembrane segment 2 (part of the α-1 repeat), is important for both Na(+) and Ca(2+) translocations. Using cysteine susceptibility analysis, we demonstrated that Ser(110) is exposed to the cytoplasm when the exchanger is in the inward state configuration.
Assuntos
Cálcio , Homeostase/fisiologia , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Sódio , Animais , Cálcio/química , Cálcio/metabolismo , Humanos , Transporte de Íons/fisiologia , Mutação , Estrutura Secundária de Proteína , Sódio/química , Sódio/metabolismo , Trocador de Sódio e Cálcio/genéticaRESUMO
The Na(+)/Ca(2+) exchanger protein was first isolated from cardiac sarcolemma in 1988 and cloned in 1990. This allowed study of Na(+)/Ca(2+) exchange at the molecular level to begin. I will review the story leading to the cloning of NCX and the research that resulted from this event. This will include structure-function studies such as determination of the numbers of transmembrane segments and topological arrangement. Information on ion transport sites has been gathered from site-directed mutagenesis. The regions involved in Ca(2+) regulation have been identified, analyzed, and crystallized.We have also generated genetically altered mice to study the role of NCX in the myocardium. Of special interest are mice with atrial- or ventricular-specific KO of NCX that reveal new information on the role of NCX in excitation-contraction coupling and in cardiac pacemaker activity.
Assuntos
Relógios Biológicos/fisiologia , Clonagem Molecular , Proteínas Musculares , Miocárdio , Sarcolema , Trocador de Sódio e Cálcio , Animais , Aniversários e Eventos Especiais , Pesquisa Biomédica/história , História do Século XX , História do Século XXI , Humanos , Transporte de Íons , Camundongos , Camundongos Transgênicos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/isolamento & purificação , Proteínas Musculares/metabolismo , Mutagênese Sítio-Dirigida , Miocárdio/química , Miocárdio/metabolismo , Estrutura Secundária de Proteína , Sarcolema/química , Sarcolema/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/isolamento & purificação , Trocador de Sódio e Cálcio/metabolismoRESUMO
Na+/Ca2+ exchangers (NCX) transport Ca2+ in or out of cells in exchange for Na+. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca2+ homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na+ and Ca2+. Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca2+ and Na+. We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. In the inward-facing state with low cytosolic [Ca2+], a TM-associated four-stranded ß-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. Ca2+ binding to the cytosolic second Ca2+-binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins.
Assuntos
Coração , Trocador de Sódio e Cálcio , Humanos , Trocador de Sódio e Cálcio/metabolismo , Transporte de Íons , Citosol/metabolismo , Cálcio/metabolismoRESUMO
Large conductance voltage and Ca(2+)-dependent K(+) channels (BK(Ca)) are activated by both membrane depolarization and intracellular Ca(2+). Recent studies on bacterial channels have proposed that a Ca(2+)-induced conformational change within specialized regulators of K(+) conductance (RCK) domains is responsible for channel gating. Each pore-forming alpha subunit of the homotetrameric BK(Ca) channel is expected to contain two intracellular RCK domains. The first RCK domain in BK(Ca) channels (RCK1) has been shown to contain residues critical for Ca(2+) sensitivity, possibly participating in the formation of a Ca(2+)-binding site. The location and structure of the second RCK domain in the BK(Ca) channel (RCK2) is still being examined, and the presence of a high-affinity Ca(2+)-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BK(Ca) and prokaryotic RCK domains that reveal the location of a second RCK domain in human BK(Ca) channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca(2+)-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca(2+)-induced change in conformation, associated with an alpha-to-beta structural transition. We also show that the Ca bowl is an essential element for the Ca(2+)-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca(2+)-dependent gating mechanism of BK(Ca) channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.
Assuntos
Cálcio/metabolismo , Canal de Potássio Kv1.6/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/química , Dicroísmo Circular , Cristalografia por Raios X/métodos , Humanos , Conformação Molecular , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrofotometria UltravioletaRESUMO
Ca2+ homeostasis is essential for cell function and survival. As such, the cytosolic Ca2+ concentration is tightly controlled by a wide number of specialized Ca2+ handling proteins. One among them is the Na+ -Ca2+ exchanger (NCX), a ubiquitous plasma membrane transporter that exploits the electrochemical gradient of Na+ to drive Ca2+ out of the cell, against its concentration gradient. In this critical role, this secondary transporter guides vital physiological processes such as Ca2+ homeostasis, muscle contraction, bone formation, and memory to name a few. Herein, we review the progress made in recent years about the structure of the mammalian NCX and how it relates to function. Particular emphasis will be given to the mammalian cardiac isoform, NCX1.1, due to the extensive studies conducted on this protein. Given the degree of conservation among the eukaryotic exchangers, the information highlighted herein will provide a foundation for our understanding of this transporter family. We will discuss gene structure, alternative splicing, topology, regulatory mechanisms, and NCX's functional role on cardiac physiology. Throughout this article, we will attempt to highlight important milestones in the field and controversial topics where future studies are required. © 2021 American Physiological Society. Compr Physiol 12:1-37, 2021.
Assuntos
Cálcio , Trocador de Sódio e Cálcio , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Coração , Homeostase , Humanos , Mamíferos/metabolismo , Proteínas de Membrana Transportadoras , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
Background Sodium-calcium (Ca2+) exchanger isoform 1 (NCX1) is the dominant Ca2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca2+ homeostasis during excitation-contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen-inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation-contraction coupling and intracellular Ca2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury. Methods and Results We used the α-myosin heavy chain promoter (Myh6)-MerCreMer promoter to create a tamoxifen-inducible cardiac-specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca2+ increased despite adaptive reductions in Ca2+ current and action potential duration and compensatory increases in excitation-contraction coupling gain, sarcoplasmic reticulum Ca2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca2+ normalized and SR Ca2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular-related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion. Conclusions Tamoxifen-inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca2+ influx, increasing alternative Ca2+ efflux pathways, and increasing excitation-contraction coupling gain to maintain contractility at the cost of mild Ca2+-activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.
Assuntos
Arritmias Cardíacas , Cálcio , Miócitos Cardíacos , Traumatismo por Reperfusão , Trocador de Sódio e Cálcio , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/prevenção & controle , Cálcio/metabolismo , Fibrose , Hipertrofia , Camundongos , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/genética , Tamoxifeno/farmacologiaRESUMO
Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.
Assuntos
Arritmias Cardíacas , Cálcio , Potenciais de Ação , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Ventrículos do Coração , Miócitos Cardíacos , Coelhos , RatosRESUMO
We expressed full-length Na(+)-Ca(2+) exchangers (NCXs) with mutations in two Ca(2+)-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca(2+)-dependent regulation of NCX. CBD1 has four Ca(2+)-binding sites, and mutation of residues Asp(421) and Glu(451), which primarily coordinate Ca(2+) at sites 1 and 2, had little effect on regulation of NCX by Ca(2+). In contrast, mutations at residues Glu(385), Asp(446), Asp(447), and Asp(500), which coordinate Ca(2+) at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca(2+). Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca(2+) affinity but retained regulation, confirming a contribution of CBD2 to Ca(2+) regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca(2+) affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca(2+)-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca(2+)-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between beta-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca(2+), indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca(2+)-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca(2+), although CBD2 is also necessary for Ca(2+)-dependent regulation.
Assuntos
Cálcio/química , Trocador de Sódio e Cálcio/química , Animais , Sítios de Ligação , Cristalografia por Raios X/métodos , Citoplasma/metabolismo , Regulação da Expressão Gênica , Íons , Mutagênese , Mutação , Oócitos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
The Na(+)-Ca(2+) exchanger plays a central role in cardiac contractility by maintaining Ca(2+) homeostasis. Two Ca(2+)-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca(2+) binding to these regulatory domains activates the transport of Ca(2+) across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca(2+) ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca(2+)-bound (1.7-A resolution) and -free (1.4-A resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca(2+) ions in primary and secondary Ca(2+) sites. In the absence of Ca(2+), Lys(585) stabilizes the structure by coordinating two acidic residues (Asp(552) and Glu(648)), one from each of the Ca(2+)-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca(2+) ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca(2+) site completely remove Ca(2+) regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na(+)-Ca(2+) exchanger.
Assuntos
Cálcio/química , Cálcio/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Sítios de Ligação , Cátions/química , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Trocador de Sódio e Cálcio/genéticaRESUMO
A precise temporal and spatial control of intracellular Ca2+ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca2+ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). NCX extrudes Ca2+ from the cell, balancing the Ca2+entering the cytoplasm during systole through L-type Ca2+ channels. In parallel, following SR Ca2+ release, SERCA activity replenishes the SR, reuptaking Ca2+ from the cytoplasm. The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca2+ potentiate NCX activity, while an increase in intracellular Na+ levels inhibits NCX via a mechanism known as Na+-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca2+, Na+ and H+ couple cell metabolism to Ca2+ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.
Assuntos
Citoplasma/metabolismo , Miocárdio/metabolismo , Prótons , Trocador de Sódio e Cálcio/metabolismo , Regulação Alostérica , Animais , Humanos , Concentração de Íons de Hidrogênio , Trocador de Sódio e Cálcio/químicaRESUMO
Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1-4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5-12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.
Assuntos
Relógios Biológicos , Cálcio/metabolismo , Acoplamento Excitação-Contração , Átrios do Coração/metabolismo , Nó Sinoatrial/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Animais , HumanosRESUMO
The cardiac Na+-Ca2+ exchanger (NCX) plays a critical role in the heart by extruding Ca2+ after each contraction and thus regulates cardiac contractility. The activity of NCX is strongly inhibited by cytosolic protons, which suggests that intracellular acidification will have important effects on heart contractility. However, the mechanisms underlying this inhibition remain elusive. It has been suggested that pH regulation originates from the competitive binding of protons to two Ca2+-binding domains within the large cytoplasmic loop of NCX and requires inactivation by intracellular Na+ to fully develop. By combining mutagenesis and electrophysiology, we demonstrate that NCX pH modulation is an allosteric mechanism distinct from Na+ and Ca2+ regulation, and we show that cytoplasmic Na+ can affect the sensitivity of NCX to protons. We further identify two histidines (His 124 and His 165) that are important for NCX proton sensitivity and show that His 165 plays the dominant role. Our results reveal a complex interplay between the different allosteric mechanisms that regulate the activity of NCX. Because of the central role of NCX in cardiac function, these findings are important for our understanding of heart pathophysiology.