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1.
Nat Commun ; 11(1): 4031, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788582

RESUMO

Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies.


Assuntos
Canais de Cálcio/genética , Evolução Molecular , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/química , Quitridiomicetos/genética , Proteínas Fúngicas/química , Células HeLa , Humanos , Funções Verossimilhança , Filogenia , Especificidade da Espécie
2.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
Nat Commun ; 11(1): 3709, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709843

RESUMO

Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.


Assuntos
Canais de Cálcio/química , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Razão Sinal-Ruído , Imagem Individual de Molécula , Fluxo de Trabalho
4.
Nature ; 582(7810): 129-133, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494073

RESUMO

Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Microscopia Crioeletrônica , Sítios de Ligação/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/ultraestrutura , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura
5.
EBioMedicine ; 52: 102646, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32062352

RESUMO

BACKGROUND: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. METHODS: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. FINDINGS: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). INTERPRETATION: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. FUNDING: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Canais de Cálcio/genética , Amplificação de Genes , Animais , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Metástase Neoplásica , Estadiamento de Neoplasias , Domínios e Motivos de Interação entre Proteínas
6.
Sci Rep ; 10(1): 2835, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071363

RESUMO

Mitochondrial Ca2+ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca2+ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca2+ dynamics, which are assumed to be determined by distinct thresholds of Ca2+ increases at each subcellular mitochondrial domain. The balance between Ca2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca2+ affinities are engineered to match the intramitochondrial Ca2+ concentrations. They enable visualization of mitochondrial Ca2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca2+ dynamics and their functions.


Assuntos
Canais de Cálcio/genética , Cálcio/metabolismo , Proteínas Luminescentes/genética , Mitocôndrias/metabolismo , Animais , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Sinalização do Cálcio/genética , Humanos , Proteínas Luminescentes/química , Dinâmica Mitocondrial/genética , Membranas Mitocondriais/metabolismo
7.
Nat Struct Mol Biol ; 27(2): 150-159, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31988524

RESUMO

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.


Assuntos
Proteínas Aviárias/metabolismo , Canais de Cálcio/metabolismo , Galinhas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Cálcio/metabolismo , Canais de Cálcio/química , Microscopia Crioeletrônica , Homeostase , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
9.
Acta Crystallogr D Struct Biol ; 76(Pt 1): 41-50, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31909742

RESUMO

Three-dimensional structure models refined using low-resolution data from crystallographic or electron cryo-microscopy experiments can benefit from high-quality restraints derived from quantum-chemical methods. However, nonperiodic atom-centered quantum-chemistry codes do not inherently account for nearest-neighbor interactions of crystallographic symmetry-related copies in a satisfactory way. Here, these nearest-neighbor effects have been included in the model by expanding to a super-cell and then truncating the super-cell to only include residues from neighboring cells that are interacting with the asymmetric unit. In this way, the fragmentation approach can adequately and efficiently include nearest-neighbor effects. It has previously been shown that a moderately sized X-ray structure can be treated using quantum methods if a fragmentation approach is applied. In this study, a target protein (PDB entry 4gif) was partitioned into a number of large fragments. The use of large fragments (typically hundreds of atoms) is tractable when a GPU-based package such as TeraChem is employed or cheaper (semi-empirical) methods are used. The QM calculations were run at the HF-D3/6-31G level. The models refined using a recently developed semi-empirical method (GFN2-xTB) were compared and contrasted. To validate the refinement procedure for a non-P1 structure, a standard set of crystallographic metrics were used. The robustness of the implementation is shown by refining 13 additional protein models across multiple space groups and a summary of the refinement metrics is presented.


Assuntos
Canais de Cálcio/química , Cristalografia por Raios X/métodos , Modelos Moleculares , Receptores de Superfície Celular/química , Software , Conformação Proteica
10.
Annu Rev Pharmacol Toxicol ; 60: 133-154, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31537174

RESUMO

Voltage-gated sodium and calcium channels are evolutionarily related transmembrane signaling proteins that initiate action potentials, neurotransmission, excitation-contraction coupling, and other physiological processes. Genetic or acquired dysfunction of these proteins causes numerous diseases, termed channelopathies, and sodium and calcium channels are the molecular targets for several major classes of drugs. Recent advances in the structural biology of these proteins using X-ray crystallography and cryo-electron microscopy have given new insights into the molecular basis for their function and pharmacology. Here we review this recent literature and integrate findings on sodium and calcium channels to reveal the structural basis for their voltage-dependent activation, fast and slow inactivation, ion conductance and selectivity, and complex pharmacology at the atomic level. We conclude with the theme that new understanding of the diseases and therapeutics of these channels will be derived from application of the emerging structural principles from these recent structural analyses.


Assuntos
Canais de Cálcio/efeitos dos fármacos , Canalopatias/tratamento farmacológico , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Canalopatias/fisiopatologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Terapia de Alvo Molecular , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo
11.
J Ethnopharmacol ; 248: 112321, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31655146

RESUMO

ETHNOPHARMACOLOGY RELEVANCE: In traditional Mexican medicine, Echeveria gibbiflora DC has been used as a vaginal post-coital rinse to prevent pregnancy. The aqueous crude extract (OBACE) induces sperm immobilization/agglutination and a hypotonic-like effect, likely attributed to the high concentration of calcium bis-(hydrogen-1-malate) hexahydrate [Ca2+ (C4H5O5)2•6H2O]. Likewise, OBACE impedes the increase of [Ca2+]i during capacitation. AIM OF THE STUDY: Evaluate the effect of OBACE on sperm energy metabolism and the underlying mechanism of action on sperm-specific channel. MATERIAL AND METHODS: In vitro, we quantified the mouse sperm immobilization effect and the antifertility potential of OBACE. The energetic metabolism status was also evaluated by assessing the ATP levels, general mitochondrial activity, mitochondrial membrane potential, and enzymatic activity of three key enzymes of energy metabolism. Furthermore, the effect of the ion efflux of Cl- and K+, as well as the pHi, were investigated in order to elucidate which channel is suitable to perform an in silico study. RESULTS: Total and progressive motility notably decreased, as did fertility rates. ATP levels, mitochondrial activity and membrane potential were reduced. Furthermore, the activities of the three enzymes decreased. Neither Cl- or K+ channels activities were affected at low concentrations of OBACE; nevertheless, pHi did not alkalinize. Finally, an in silico analysis was performed between the Catsper channel and calcium bis-(hydrogen-1-malate) hexahydrate, which showed a possible blockade of this sperm cation channel. CONCLUSION: The results were useful to elucidate the effect of OBACE and to propose it as a future male contraceptive.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Anticoncepcionais Masculinos/farmacologia , Crassulaceae , Metabolismo Energético/efeitos dos fármacos , Extratos Vegetais/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Sítios de Ligação , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Anticoncepcionais Masculinos/química , Anticoncepcionais Masculinos/isolamento & purificação , Crassulaceae/química , Fertilidade/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Extratos Vegetais/isolamento & purificação , Conformação Proteica , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/metabolismo , Relação Estrutura-Atividade
12.
Cell Physiol Biochem ; 53(S1): 11-43, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31834993

RESUMO

Ion channels residing in the inner (IMM) and outer (OMM) mitochondrial membranes are emerging as noteworthy pharmacological targets in oncology. While these aspects have not been investigated for all of them, a role in cancer growth and/or metastasis and/or drug resistance has been shown at least for the IMM-residing Ca2+ uniporter complex and K+- selective mtKV1.3, mtIKCa, mtSKCa and mtTASK-3, and for the OMM Voltage-Dependent Anion Channel (mitochondrial porin). A special case is that of the Mitochondrial Permeability Transition Pore, a large pore which forms in the IMM of severely stressed cells, and which may be exploited to precipitate the death of cancerous cells. Here we briefly discuss the oncological relevance of mitochondria and their channels, and summarize the methods that can be adopted to selectively target these intracellular organelles. We then present an updated list of known mitochondrial channels, and review the pharmacology of those with proven relevance for cancer.


Assuntos
Antineoplásicos/química , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Bibliotecas de Moléculas Pequenas/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Humanos , Canais Iônicos/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Canais de Potássio/química , Canais de Potássio/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/metabolismo
13.
Cell Physiol Biochem ; 53(S1): 44-51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31834994

RESUMO

The current basis of systemic treatment for pancreas cancer involves cytotoxic chemotherapy. Despite newer regimens, overall survival remains poor and this dilemma is further compounded by a lack of effective, novel therapeutic targets. Another challenge in treating pancreas cancer is the complex tumor microenvironment, which contains regulatory T cells, myeloid derived suppressor cells, and tumor associated macrophages that forms a barrier to standard therapies. Intracellular ion channels are ubiquitously expressed in all cells and their role in carcinogenesis is increasingly becoming elucidated. They play an integral role in each of the six "Hallmarks of Cancer" and are potential novel prognostic biomarkers and therapeutic targets for pancreas cancer. Although examined in various hematologic and gastrointestinal malignancies, there are limited data examining the prognostic role of specific ion channels in pancreas ductal adenocarcinoma. This review focuses on chloride (CLCA-1, CLIC1, CLIC3), calcium (TRPM7, TRPM8), and potassium (Kir3.1, KCa3.1, Kv11.1, Kv1.3) channels in pancreas cancer.


Assuntos
Canais Iônicos/metabolismo , Neoplasias Pancreáticas/patologia , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Ensaios Clínicos como Assunto , Humanos , Canais Iônicos/química , Neoplasias Pancreáticas/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Prognóstico , Microambiente Tumoral
14.
Proc Natl Acad Sci U S A ; 116(51): 26008-26019, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796582

RESUMO

The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1-PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure-activity studies, thereby suggesting strategies for rational drug design.


Assuntos
Simulação de Acoplamento Molecular , Piperidinas/farmacologia , Canal de Cátion TRPA1/química , Canal de Cátion TRPA1/efeitos dos fármacos , Animais , Sítios de Ligação , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Desenho de Fármacos , Humanos , Isotiocianatos , Ligantes , Modelos Estruturais , Mutagênese , Oximas/farmacologia , Propofol/farmacologia , Domínios Proteicos , Ratos , Especificidade da Espécie , Canal de Cátion TRPA1/metabolismo
15.
Elife ; 82019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31868580

RESUMO

Human-based modelling and simulations are becoming ubiquitous in biomedical science due to their ability to augment experimental and clinical investigations. Cardiac electrophysiology is one of the most advanced areas, with cardiac modelling and simulation being considered for virtual testing of pharmacological therapies and medical devices. Current models present inconsistencies with experimental data, which limit further progress. In this study, we present the design, development, calibration and independent validation of a human-based ventricular model (ToR-ORd) for simulations of electrophysiology and excitation-contraction coupling, from ionic to whole-organ dynamics, including the electrocardiogram. Validation based on substantial multiscale simulations supports the credibility of the ToR-ORd model under healthy and key disease conditions, as well as drug blockade. In addition, the process uncovers new theoretical insights into the biophysical properties of the L-type calcium current, which are critical for sodium and calcium dynamics. These insights enable the reformulation of L-type calcium current, as well as replacement of the hERG current model.


Assuntos
Potenciais de Ação/fisiologia , Modelos Cardiovasculares , Miócitos Cardíacos , Algoritmos , Biofísica , Cálcio/química , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Calibragem , Simulação por Computador , Eletrocardiografia , Fenômenos Eletrofisiológicos , Eletrofisiologia , Acoplamento Excitação-Contração , Cardiopatias/fisiopatologia , Ventrículos do Coração/patologia , Humanos , Sódio/química , Sódio/metabolismo
16.
Proc Natl Acad Sci U S A ; 116(48): 24359-24365, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719194

RESUMO

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.


Assuntos
Metionina/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cloraminas/química , Escherichia coli/genética , Temperatura Alta , Humanos , Peróxido de Hidrogênio/química , Macrófagos , Metionina/química , Mutação , Oxidantes/química , Oxirredução , Técnicas de Patch-Clamp , Fagocitose , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Compostos de Tosil/química
17.
Int J Mol Sci ; 20(19)2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569359

RESUMO

Calcium ions (Ca2+) influx to mitochondrial matrix is crucial for the life of a cell. Mitochondrial calcium uniporter (mtCU) is a protein complex which consists of the pore-forming subunit (MCU) and several regulatory subunits. MtCU is the main contributor to inward Ca2+ currents through the inner mitochondrial membrane. Extensive investigations of mtCU involvement into normal and pathological molecular pathways started from the moment of discovery of its molecular components. A crucial role of mtCU in the control of these pathways is now recognized in both health and disease. In particular, impairments of mtCU function have been demonstrated for cardiovascular and skeletal muscle-associated pathologies. This review summarizes the current state of knowledge on mtCU structure, regulation, and function in different types of muscle tissues in health and disease.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Suscetibilidade a Doenças , Músculos/metabolismo , Animais , Cálcio/química , Cálcio/metabolismo , Sinalização do Cálcio , Fenômenos Eletrofisiológicos , Humanos , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Miocárdio/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
18.
Int J Oncol ; 55(5): 1090-1096, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545410

RESUMO

Uveal malignant melanoma (UMM), the most common primary adult intraocular tumor with a marked metastatic potential, is genetically unique and has unfortunately had few treatment breakthroughs. In this study, we subjected a UMM cell line to high­throughput library screening with 1,018 FDA­approved compounds to identify potential UMM­selective cytotoxic agents. Amlodipine, a dihydropyridine calcium channel blocker (CCB), ranked no. 2 and no. 8 of the most cytotoxic compounds. Thus, we further characterized the differential effects of calcium blockade on UMM and cutaneous malignant melanoma (CMM) lines in vitro using growth inhibition, cell cycle progression, apoptosis and senescence assays. Amlodipine had a significantly higher growth inhibitory potency in UMM (IC50=13.1 µM) than CMM (IC50=15.9 µM, P<0.05) lines. In 3D spherical cell culture, amlodipine treatment significantly impaired tissue volume growth in two UMM lines, but exerted no significant effects among all 3 CMM lines tested. Treatment with 10 and 20 µM amlodipine induced a significant impairment of cell cycle progression and the apoptosis of UMM lines, implicating both of these processes as mediators of the observed growth inhibition in UMM compared to CMM. On the whole, the findings of this study suggest that calcium channel blockade is a potentially effective strategy for selective uveal melanoma targeting.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Cálcio/metabolismo , Melanoma/tratamento farmacológico , Neoplasias Uveais/tratamento farmacológico , Apoptose , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Ciclo Celular , Movimento Celular , Proliferação de Células , Ensaios de Triagem em Larga Escala , Humanos , Melanoma/metabolismo , Melanoma/patologia , Células Tumorais Cultivadas , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia
19.
Mol Pharmacol ; 96(4): 485-492, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31391290

RESUMO

Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+ Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel-blocking drugs at the atomic level. SIGNIFICANCE STATEMENT: Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Diltiazem/farmacologia , Regulação Alostérica , Anlodipino/química , Anlodipino/farmacologia , Animais , Sítios de Ligação , Bloqueadores dos Canais de Cálcio/química , Cristalografia por Raios X , Diltiazem/química , Humanos , Modelos Moleculares , Conformação Proteica , Verapamil/farmacologia
20.
EMBO Rep ; 20(9): e47488, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31397067

RESUMO

The mitochondrial calcium uniporter (MCU) complex mediates the uptake of Ca2+ into mitochondria. Its activity is regulated by a heterodimer of MICU1 and MICU2, two EF-hand-containing proteins that act as the main gatekeeper of the uniporter. Herein we report the crystal structure of human MICU2 at 1.96 Å resolution. Our structure reveals a dimeric architecture of MICU2, in which each monomer adopts the canonical two-lobe structure with a pair of EF-hands in each lobe. Both Ca2+ -bound and Ca2+ -free EF-hands are observed in our structure. Moreover, we characterize the interaction sites within the MICU2 homodimer, as well as the MICU1-MICU2 heterodimer in both Ca2+ -free and Ca2+ -bound conditions. Glu242 in MICU1 and Arg352 in MICU2 are crucial for apo heterodimer formation, while Phe383 in MICU1 and Glu196 in MICU2 significantly contribute to the interaction in the Ca2+ -bound state. Based on our structural and biochemical analyses, we propose a model for MICU1-MICU2 heterodimer formation and its conformational transition from apo to a more compact Ca2+ -bound state, which expands our understanding of this co-regulatory mechanism critical for MCU's mitochondrial calcium uptake function.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Dimerização , Humanos , Modelos Biológicos , Ligação Proteica
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