RESUMO
The skeletal muscle dihydropyridine receptor (DHPR) ß1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitationcontraction (EC) coupling. Previously, we showed that all four vertebrate ß isoforms (ß1ß4) facilitate α1S triad targeting and, except for ß3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 74887493 (2013)]. Consequently, ß3 failed to restore EC coupling despite the fact that both ß3 and ß1a restore tetrads. Thus, all ß-subunits are able to restore triad targeting, but only ß1a restores both tetrads and proper DHPRRyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 74887493 (2013)]. To investigate the molecular region(s) of ß1a responsible for the tetradic arrangement of DHPRs and thus DHPRRyR1 coupling, we expressed loss- and gain-of-function chimeras between ß1a and ß4, with systematically swapped domains in zebrafish strain relaxed (ß1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. ß1a/ß4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from ß4 showed complete restoration of SR Ca2+ release. However, chimera ß1a/ß4(C) with ß4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera ß4/ß1a(C) with ß1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of ß1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPRRyR1 interaction, essential for skeletal muscle EC coupling.
Assuntos
Canais de Cálcio Tipo L , Fibras Musculares Esqueléticas , Canal de Liberação de Cálcio do Receptor de Rianodina , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Acoplamento Excitação-Contração , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The dihydropyridine receptor (DHPR) ß1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform ß3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in ß1-null zebrafish relaxed myotubes, unlike the other three vertebrate ß-isoforms (ß1a, ß2a, and ß4). Thus, we used ß3 for chimerization with ß1a to investigate whether any of the five distinct molecular regions of ß1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between ß1a and ß3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of ß1a in charge movement restoration. More interestingly, ß1a SH3 domain and C terminus, when simultaneously engineered into ß3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of ß1a (also of ß2a and ß4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which ß subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Domínios de Homologia de src , Motivos de Aminoácidos , Animais , Acoplamento Excitação-Contração , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca(2+) channel (Ca(V)1.1). Ca(V)1.1 in turn triggers opening of the sarcoplasmic Ca(2+) release channel (RyR1) via interchannel protein-protein interaction to release Ca(2+) for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca(2+) inward current through Ca(V)1.1 is found in mammalian skeletal myotubes. The role of this Ca(2+) influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca(2+) currents. We identified two distinct isoforms of the pore-forming Ca(V)1.1alpha(1S) subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca(2+) but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca(2+) conductivity of both Ca(V)1.1alpha(1S) isoforms is a common trait of all higher teleosts. This non-Ca(2+) conductivity of Ca(V)1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca(2+), it evolved toward Ca(V)1.1-RyR1 protein-protein interaction with a relatively small and slow influx of external Ca(2+) in tetrapods. Finally, the Ca(V)1.1 Ca(2+) influx was completely eliminated in higher teleost fishes.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Peixes/fisiologia , Músculo Esquelético/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Evolução Molecular , Acoplamento Excitação-Contração , Peixes/genética , Técnicas In Vitro , Dados de Sequência Molecular , Contração Muscular/fisiologia , Técnicas de Patch-Clamp , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição Tecidual , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (KATP ) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV 1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.
Assuntos
Doenças Musculares , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Camundongos , Trifosfato de Adenosina , Músculo Esquelético/metabolismo , Doenças Musculares/induzido quimicamente , Doenças Musculares/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Verapamil/metabolismoRESUMO
Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.
Assuntos
Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Músculo Esquelético/metabolismo , Mutação Puntual , Animais , Células Cultivadas , Acoplamento Excitação-Contração , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Permeabilidade , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to -25 to -35 mV in the genetic and pharmacologic models of Becker disease. Both Na+ and Ca2+ currents contribute to plateau potentials. Na+ persistent inward current (NaPIC) through NaV1.4 channels is the key trigger of plateau potentials and current through CaV1.1 Ca2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita.
Myotonia is a neuromuscular condition that causes problems with the relaxation of muscles following voluntary movements. One type of myotonia is Becker disease, also called recessive myotonia congenita. This is a genetic condition that causes muscle stiffness as a result of involuntary muscle activity. Patients may also suffer transient weakness for a few seconds or as long as several minutes after initiating a movement. The cause of these bouts of temporary weakness is still unclear, but there are hints that it could be linked to the muscle losing its excitability, the ability to respond to the stimuli that make it contract. However, this is at odds with findings that show that muscles in Becker disease are hyperexcitable. Muscle excitability depends on the presence of different concentrations of charged ions (positively charged sodium, calcium and potassium ions and negatively charged chloride ions) inside and outside of each muscle cells. These different concentrations of ions create an electric potential across the cell membrane, also called the 'membrane potential'. When a muscle cell gets stimulated, proteins on the cell membrane known as ion channels open. This allows the flow of ions between the inside and the outside of the cell, which causes an electrical current that triggers muscle contraction. To better understand the causes behind this muscle weakness, Myers et al. used mice that had either been genetically manipulated or given drugs to mimic Becker disease. By measuring both muscle force and the electrical currents that drive contraction, Myers et al. found that the mechanism underlying post-movement weakness involved a transient change in the concentrations of positively charged ions inside and outside the cells. Further experiments showed that proteins that regulate the passage of both sodium and calcium in and out of the cell called sodium and calcium channels contributed to this change in concentration. In addition, Myers et al. discovered that using a drug called ranolazine to stop sodium ions from entering the cell eliminated transient weakness in live mice. These findings suggest that in Becker disease, muscles cycle rapidly between being hyperexcited or not able to be excited, and that targeting the flow of sodium ions into the cell could be an effective treatment to prevent transient weakness in myotonia congenita. This study paves the way towards the development of new therapies to treat Becker disease as well as other muscle ion channel diseases with transient weakness such as periodic paralysis.
Assuntos
Potenciais da Membrana/fisiologia , Miotonia Congênita/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Miotonia Congênita/diagnóstico , Miotonia Congênita/genética , Sódio/fisiologiaRESUMO
To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca(2+) channel (Ca(V)) α(1S)-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of Ca(V)α(1S) in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged α(1S)T1354S in dysgenic (α(1S)-null) myotubes. Whole cell patch-clamp analysis revealed that α(1S)T1354S produced significantly faster activation of L-type Ca(2+) currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α(1S) (α(1S)WT). In addition, α(1S)T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca(2+) release and to larger action-potential-induced intracellular Ca(2+) transients under low (≤ 2 mM) caffeine concentrations compared with α(1S)WT. Thus our data suggest that an additional influx of Ca(2+) due to faster activation of the α(1S)T1354S L-type Ca(2+) current, in concert with higher caffeine sensitivity of Ca(2+) release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.
Assuntos
Canais de Cálcio/genética , Hipertermia Maligna/genética , Mutação Puntual , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Cafeína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Canais de Cálcio Tipo L , Células Cultivadas , Acoplamento Excitação-Contração/efeitos dos fármacos , Acoplamento Excitação-Contração/fisiologia , Feminino , Ligação Genética , Loci Gênicos , Humanos , Masculino , Hipertermia Maligna/fisiopatologia , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , CoelhosRESUMO
In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Músculo Esquelético/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/química , Ativação do Canal Iônico , Camundongos Endogâmicos C57BL , Modelos Animais , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Nifedipino/farmacologiaRESUMO
The Ca2+-activated Cl- channel (CaCC) TMEM16A/Anoctamin 1 (ANO1) is expressed in gastrointestinal epithelia and smooth muscle cells where it mediates secretion and intestinal motility. However, ANO1 Cl- conductance has never been reported to play a role in skeletal muscle. Here we show that ANO1 is robustly expressed in the highly evolved skeletal musculature of the euteleost species zebrafish. We characterised ANO1 as bonafide CaCC which is activated close to maximum by Ca2+ ions released from the SR during excitation-contraction (EC) coupling. Consequently, our study addressed the question about the physiological advantage of implementation of ANO1 into the euteleost skeletal-muscle EC coupling machinery. Our results reveal that Cl- influx through ANO1 plays an essential role in restricting the width of skeletal-muscle action potentials (APs) by accelerating the repolarisation phase. Resulting slimmer APs enable higher AP-frequencies and apparently tighter controlled, faster and stronger muscle contractions, crucial for high speed movements.
Assuntos
Potenciais de Ação/fisiologia , Anoctamina-1/metabolismo , Cálcio/metabolismo , Músculo Esquelético/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Anoctamina-1/genética , Cloretos/metabolismo , Humanos , Modelos Biológicos , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Retículo Sarcoplasmático/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (CaV1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth.
Assuntos
Cálcio/metabolismo , Junção Neuromuscular/fisiologia , Crescimento Neuronal/fisiologia , Receptores Colinérgicos/metabolismo , Animais , Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Diafragma/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Fetal , Camundongos , Camundongos Knockout , Neurônios Motores/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/deficiência , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein-protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR alpha(1S) subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (alpha(1S)-null) myotubes reconstituted with distinct alpha(1) subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that alpha(1S) and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, alpha(1C) and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an alpha(1S)-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Membrana Celular/metabolismo , Técnica de Fratura por Congelamento , Moscas Domésticas , Tamanho da Partícula , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The L-type Ca2+ channel or dihydropyridine receptor (DHPR) in vertebrate skeletal muscle is responsible for sensing sarcolemmal depolarizations and transducing this signal to the sarcoplasmic Ca2+ release channel RyR1 via conformational coupling to initiate muscle contraction. During this excitation-contraction (EC) coupling process there is a slow Ca2+ current through the mammalian DHPR which is fully missing in euteleost fishes. In contrast to ancestral evolutionary stages where skeletal muscle EC coupling is still depended on Ca2+-induced Ca2+-release (CICR), it is possible that the DHPR Ca2+ conductivity during mammalian (conformational) EC coupling was retained as an evolutionary remnant (vestigiality). Here, we wanted to test the hypothesis that due to the lack of evolutionary pressure in post-CICR species skeletal muscle DHPR Ca2+ conductivity gradually reduced as evolution progressed. Interestingly, we identified that the DHPR of the early ray-finned fish sterlet (Acipenser ruthenus) is phylogenetically positioned above the mammalian rabbit DHPR which retained robust Ca2+ conductivity, but below the euteleost zebrafish DHPR which completely lost Ca2+ conductivity. Remarkably, our results revealed that sterlet DHPR still retained the Ca2+ conductivity but currents are significantly reduced compared to rabbit. This decrease is due to lower DHPR membrane expression similar to zebrafish, as well as due to reduced channel open probability (Po). In both these fish species the lower DHPR expression density is partially compensated by higher efficacy of DHPR-RyR1 coupling. The complete loss of Po in zebrafish and other euteleost species was presumably based on the teleost specific 3rd round of genome duplication (Ts3R). Ts3R headed into the appearance of two skeletal muscle DHPR isoforms which finally, together with the radiation of the euteleost clade, fully lost the Po.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Peixes , Músculo Esquelético/metabolismo , Filogenia , Animais , Células Cultivadas , Camundongos , Coelhos , Peixe-ZebraRESUMO
Skeletal muscle excitation-contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and L-type Ca2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca2+ channel is still enigmatic. Here we show that Ca2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca2+ influx is eliminated. Homozygous knock-in mice display SR Ca2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca2+ influx in mammalian skeletal muscle is an evolutionary remnant.In mammalian skeletal muscle, the DHPR functions as a voltage sensor to trigger muscle contraction and as a Ca2+ channel. Here the authors show that mice where Ca2+ influx through the DHPR is eliminated display no difference in skeletal muscle function, suggesting that the Ca2+ influx through this channel is vestigial.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Músculo Esquelético/fisiologia , Animais , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Retículo Sarcoplasmático/metabolismoRESUMO
Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an â¼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Ativação do Canal Iônico , Potenciais da Membrana , Motivos de Aminoácidos , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Linhagem Celular , Humanos , Camundongos , Mioblastos/metabolismo , Mioblastos/fisiologia , Domínios Proteicos , Proteínas RecombinantesRESUMO
The neuronal L-type calcium channels (LTCCs) Cav1.2alpha1 and Cav1.3alpha1 are functionally distinct. Cav1.3alpha1 activates at lower voltages and inactivates more slowly than Cav1.2alpha1, making it suitable to support sustained L-type Ca2+ inward currents (ICa,L) and serve in pacemaker functions. We compared the biophysical and pharmacological properties of human retinal Cav1.4alpha1 using the whole-cell patch-clamp technique after heterologous expression in tsA-201 cells with other L-type alpha1 subunits. Cav1.4alpha1-mediated inward Ba2+ currents (IBa) required the coexpression of alpha2delta1 and beta3 or beta2a subunits and were detected in a lower proportion of transfected cells than Cav1.3alpha1. IBa activated at more negative voltages (5% activation threshold; -39mV; 15 mm Ba2+) than Cav1.2alpha1 and slightly more positive than Cav1.3alpha1. Voltage-dependent inactivation of IBa was slower than for Cav1.2alpha1 and Cav1.3alpha1( approximately 50% inactivation after 5 sec; alpha2delta1 + beta3 coexpression). Inactivation was not increased with Ca2+ as the charge carrier, indicating the absence of Ca2+-dependent inactivation. Cav1.4alpha1 exhibited voltage-dependent, G-protein-independent facilitation by strong depolarizing pulses. The dihydropyridine (DHP)-antagonist isradipine blocked Cav1.4alpha1 with approximately 15-fold lower sensitivity than Cav1.2alpha1 and in a voltage-dependent manner. Strong stimulation by the DHP BayK 8644 was found despite the substitution of an otherwise L-type channel-specific tyrosine residue in position 1414 (repeat IVS6) by a phenylalanine. Cav1.4alpha1 + alpha2delta1 + beta channel complexes can form LTCCs with intermediate DHP antagonist sensitivity lacking Ca2+-dependent inactivation. Their biophysical properties should enable them to contribute to sustained ICa,L at negative potentials, such as required for tonic neurotransmitter release in sensory cells and plateau potentials in spiking neurons.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Di-Hidropiridinas/farmacologia , Ativação do Canal Iônico/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Sequência de Aminoácidos , Bário/metabolismo , Cálcio/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Linhagem Celular , Clonagem Molecular , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Isradipino/farmacologia , Rim/citologia , Rim/metabolismo , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
In skeletal muscle excitation-contraction (EC) coupling the sarcolemmal L-type Ca(2+) channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca(2+) release channel RyR1 via protein-protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRalpha(1S) subunit is essential for this process, the intracellular DHPRbeta(1a) subunit was also shown to be indispensable. We previously found that the beta(1a) subunit is essential to target the DHPR into groups of four (tetrads) opposite the RyR1 homotetramers, a prerequisite for skeletal muscle EC coupling. Earlier, a unique hydrophobic heptad repeat motif (Lcdots, three dots, centeredVcdots, three dots, centeredV) in the C-terminus of beta(1a) was postulated by others to be essential for skeletal muscle EC coupling, as substitution of these residues with alanines resulted in 80% reduction of RyR1 Ca(2+) release. Therefore, we wanted to address the question if the proposed beta(1a) heptad repeat motif could be an active element of the DHPR-RyR1 signal transduction mechanism or already contributes at the ultrastructural level i.e. DHPR tetrad arrangement. Surprisingly, our experiments revealed full tetrad formation and an almost complete restoration of EC coupling in beta(1)-null zebrafish relaxed larvae and isolated myotubes upon expression of a beta(1a)-specific heptad repeat mutant (LVV to AAA) and thus contradict the earlier results.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Acoplamento Excitação-Contração , Músculo Esquelético/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Células Cultivadas , Técnicas de Inativação de Genes , Larva/citologia , Larva/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Alinhamento de Sequência , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) beta(1a) subunit. Lack of beta(1a) results in (i) reduced membrane expression of the pore forming DHPR alpha(1S) subunit, (ii) elimination of alpha(1S) charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of beta(1a) from rather general functions of beta isoforms. Zebrafish and mammalian beta(1a) subunits quantitatively restored alpha(1S) triad targeting and charge movement as well as intracellular Ca(2+) release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal beta(2a) as the phylogenetically closest, and the ancestral housefly beta(M) as the most distant isoform to beta(1a) also completely recovered alpha(1S) triad expression and charge movement. However, both revealed drastically impaired intracellular Ca(2+) transients and very limited tetrad formation compared with beta(1a). Consequently, larval motility was either only partially restored (beta(2a)-injected larvae) or not restored at all (beta(M)). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested beta subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the beta(1a) isoform. Consequently, we postulate a model that presents beta(1a) as an allosteric modifier of alpha(1S) conformation enabling skeletal muscle-type EC coupling.
Assuntos
Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo L/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Canais de Cálcio Tipo L/genética , Fibras Musculares Esqueléticas/metabolismo , Ligação Proteica , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Peixe-Zebra/genéticaRESUMO
In skeletal muscle, coupling between the 1,4-dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) underlies excitation-contraction (EC) coupling. The III-IV loop of the DHPR alpha(1S) subunit binds to a segment of RyR1 in vitro, and mutations in the III-IV loop alter the voltage dependence of EC coupling, raising the possibility that this loop is directly involved in signal transmission from the DHPR to RyR1. To clarify the role of the alpha(1S) III-IV loop in EC coupling, we examined the functional properties of a chimera (GFP-alpha(1S)[III-IVa]) in which the III-IV loop of the divergent alpha(1A) isoform replaced that of alpha(1S). Dysgenic myotubes expressing GFP-alpha(1S)[III-IVa] yielded myoplasmic Ca(2+) transients that activated at approximately 10 mV more hyperpolarized potentials and that were approximately 65% smaller than those of GFP-alpha(1S). A similar reduction was observed in voltage-dependent charge movements for GFP-alpha(1S)[III-IVa], indicating that the chimeric channels trafficked less well to the membrane but that those that were in the membrane functioned as efficiently in EC coupling as GFP-alpha(1S). Relative to GFP-alpha(1S), L-type currents mediated by GFP-alpha(1S)[III-IVa] were approximately 40% smaller and activated at approximately 5 mV more hyperpolarized potentials. The altered gating of GFP-alpha(1S)[III-IVa] was accentuated by exposure to +/-Bay K 8644, which caused a much larger hyperpolarizing shift in activation compared with its effect on GFP-alpha(1S). Taken together, our observations indicate that the alpha(1S) III-IV loop is not directly involved in EC coupling but does influence DHPR gating transitions important both for EC coupling and activation of L-type conductance.
Assuntos
Canais de Cálcio Tipo L/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Músculos/metabolismo , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Coelhos , Homologia de Sequência de AminoácidosRESUMO
The skeletal muscle dihydropyridine receptor is a slowly-activating calcium channel that functions as the voltage sensor in excitation-contraction coupling. In addition to the pore-forming alpha(1S) subunit it contains the transmembrane alpha(2)delta-1 and gamma(1) subunits and the cytoplasmic beta(1a) subunit. Although the roles of the auxiliary subunits in calcium channel function have been intensively studied in heterologous expression systems, their functions in excitation-contraction coupling has only recently been elucidated in muscle cells of various null-mutant animal models. In this article we will briefly outline the current state of these investigations.