RESUMO
TRPV6 calcium channel is a prospective target in prostate cancer (PCa) since it is not expressed in healthy prostate while its expression increases during cancer progression. Despite the role of TRPV6 in PCa cell survival and apoptotic resistance has been already established, no reliable tool to target TRPV6 channel in vivo and thus to reduce tumor burden is known to date. Here we report the generation of mouse monoclonal antibody mAb82 raised against extracellular epitope of the pore region of the channel. mAb82 inhibited TRPV6 currents by 90% at 24 µg/ml in a dose-dependent manner while decreasing store-operated calcium entry to 56% at only 2.4 µg/ml. mAb82 decreased PCa survival rate in vitro by 71% at 12 µg/ml via inducing cell death through the apoptosis cascade via activation of the protease calpain, following bax activation, mitochondria enlargement, and loss of cristae, Cyt C release, pro-caspase 9 cleavage with the subsequent activation of caspases 3/7. In vivo, mice bearing either PC3Mtrpv6+/+ or PC3Mtrpv6-/-+pTRPV6 tumors were successfully treated with mAb82 at the dose as low as 100 µg/kg resulting in a significant reduction tumor growth by 31% and 90%, respectively. The survival rate was markedly improved by 3.5 times in mice treated with mAb82 in PC3Mtrpv6+/+ tumor group and completely restored in PC3Mtrpv6-/-+pTRPV6 tumor group. mAb82 showed a TRPV6-expression dependent organ distribution and virtually no toxicity in the same way as mAbAU1, a control antibody of the same Ig2a isotype. Overall, our data demonstrate for the first time the use of an anti-TRPV6 monoclonal antibody in vitro and in vivo in the treatment of the TRPV6-expressing PCa tumors.
Assuntos
Anticorpos Monoclonais , Apoptose , Canais de Cálcio , Neoplasias da Próstata , Canais de Cátion TRPV , Masculino , Canais de Cátion TRPV/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Apoptose/efeitos dos fármacos , Humanos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Camundongos , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Calpaína/metabolismo , Cálcio/metabolismoRESUMO
There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.
Assuntos
Neoplasias da Próstata , Sódio , Masculino , Humanos , Sódio/metabolismo , Canais Iônicos/metabolismo , Transporte de Íons , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against extracellular epitopes corresponding to the extracellular loop between S1 and S2 (rb79) and the pore region (rb82). These antibodies generated a complex biphasic response with the transient activation of the TRPV6 channel. Store-operated calcium entry was consequently potentiated in the prostate cancer cell line LNCaP upon the treatment. Both rb79 and rb82 antibodies significantly decreased cell survival rate in a dose-dependent manner as compared to the control antibodies of the same isotype. This decrease was due to the enhanced cell death via apoptosis revealed using a sub-G1 peak in a cell cycle assay, TUNEL assay, and a Hoechst staining, having no effects in the PC3Mtrpv6-/- cell line. Moreover, all TUNEL-positive cells had TRPV6 membrane staining as compared to the control antibody treatment where TRPV6-positive cells were all TUNEL negative. These data clearly demonstrate that TRPV6 channel targeting using rb79 and rb82 antibodies is fatal and may be successfully used in the anticancer therapies.
RESUMO
Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftr gene in vivo in a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTR gene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.
Assuntos
Códon sem Sentido , Fibrose Cística , Camundongos , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Códon de Terminação/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genéticaRESUMO
Aim: Inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitous calcium (Ca2+) channel involved in the regulation of cellular fate and motility. Its modulation by anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) plays an important role in cancer progression. Disrupting this interaction could overcome apoptosis avoidance, one of the hallmarks of cancer, and is, thus, of great interest. Earlier reports have shown the involvement of both the Bcl-2 homology 4 (BH4) and the transmembrane domains (TMDs) of Bcl-2 in regulating IP3R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IP3R-independent action at the mitochondria (Oncotarget. 2016;7:55704-20. doi: 10.18632/oncotarget.11005). The aim of this study was to investigate how targeting the BH3 hydrophobic cleft of Bcl-2 affects IP3R:Bcl-2 interaction. Methods: Organelle membrane-derived (OMD) patch-clamp and circular dichroism (CD) thermal melting experiments were used to elucidate the effects of the ABT-199 (venetoclax) on the IP3R:Bcl-2 interaction. Molecular dynamics (MD) simulations of free and ABT-199 bound Bcl-2 were used to propose a molecular model of such interaction. Results: It was shown that occlusion of Bcl-2's hydrophobic cleft by the drug ABT-199 finely modulates IP3R gating in the low open probability (Po) regime, characteristic of the basal IP3R activity in non-excited cells. Complementary MD simulations allowed to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2. Conclusions: Bcl-2 is an important regulator of IP3R activity and, thus of Ca2+ release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Thus, it was found that the occlusion of the hydrophobic cleft of Bcl-2 by ABT-199 disrupts IP3R activity, leading to Bcl-2 rebinding with smaller affinity and lesser inhibitory effect. MDs simulations of free and ABT-199 bound Bcl-2 propose a molecular model of such disruption, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.
RESUMO
Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.
Assuntos
Carcinogênese/patologia , Neoplasias/patologia , Canais de Cátion TRPC/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Cultura Primária de CélulasRESUMO
In recent decades cancer emerged as one of the leading causes of death in the developed countries, with some types of cancer contributing to the top 10 causes of death on the list of the World Health Organization. Carcinogenesis, a malignant transformation causing formation of tumors in normal tissues, is associated with changes in the cell cycle caused by suppression of signaling pathways leading to cell death and facilitation of those enhancing proliferation. Further progression of cancer, during which benign tumors acquire more aggressive phenotypes, is characterized by metastatic dissemination through the body driven by augmented motility and invasiveness of cancer cells. All these processes are associated with alterations in calcium homeostasis in cancer cells, which promote their proliferation, motility and invasion, and dissuade cell death or cell cycle arrest. Remodeling of store-operated calcium entry (SOCE), one of the major pathways regulating intracellular Ca2+ concentration ([Ca2+]i), manifests a key event in many of these processes. This review systematizes current knowledge on the mechanisms recruiting SOCE-related proteins in carcinogenesis and cancer progression.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Carcinogênese/metabolismo , Neoplasias/metabolismo , Animais , Humanos , Neoplasias/patologiaRESUMO
Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Neoplasias da Próstata/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Idoso , Processamento Alternativo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Próstata/citologia , Próstata/metabolismo , Neoplasias da Próstata/genética , Domínios Proteicos , Canais de Cátion TRPM/químicaRESUMO
Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp "golden standard", is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.
Assuntos
Fracionamento Celular/métodos , Membranas Intracelulares , Organelas , Linhagem Celular Tumoral , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Store-operated calcium entry (SOCE) plays important roles in a multitude of cellular processes, from muscle contraction to cellular proliferation and migration. Dysregulation of SOCE is responsible for the advancement of multiple diseases, ranging from immune diseases, myopathies, to terminal ones like cancer. Naturally, SOCE has been a focus of many studies and review papers which, however, primarily concentrated on the principal players localized to the plasma membrane and responsible for Ca2+ entry into the cell. Much less has been said about other players participating in the entire SOCE event. This review aims to address this shortcoming by discussing the accumulated scientific knowledge focused on the inositol trisphosphate receptors (IP3Rs), principal player responsible for emptying intracellular Ca2+ stores in a majority of cells, and their involvement in regulation of cell migration and invasion in cancer.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Movimento Celular/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Doenças Metabólicas/metabolismo , Neoplasias/metabolismo , Animais , HumanosRESUMO
The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2ß, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cálcio/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Receptores de Inositol 1,4,5-Trifosfato/química , Domínios Proteicos , Sulfonamidas/farmacologiaRESUMO
Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Apoptose/genética , Cálcio/metabolismo , Movimento Celular/genética , Proliferação de Células , Expressão Gênica , Humanos , Família Multigênica , Neoplasias/patologia , Transdução de SinaisRESUMO
Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Temperatura Baixa , Epiderme/metabolismo , Queratinócitos/citologia , Isoformas de Proteínas/fisiologia , Canais de Cátion TRPM/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Superóxidos/metabolismoRESUMO
Transient Receptor Potential (TRP) channel proteins are a diverse family of proteins that are expressed in many organisms, tissues and cell types. TRP channels respond to a variety of stimuli, including light, mechanical or chemical stimuli, temperature, pH or osmolarity. In addition, several TRP family members have been identified as downstream molecules in the G protein-coupled receptor signaling pathway. TRP proteins are involved in a variety of cell functions both in non-excitable and excitable cells due to their diverse permeability to cations and their ability to modulate intracellular Ca2+ signaling. Emerging evidence suggests that TRP channel dysfunction significantly contributes to the physiopathology of a number of diseases, including cardiovascular, neurological, metabolic or neoplastic disorders. This review focuses on the implication of TRP proteins in the pathogenesis of some of the most prevalent disorders in human. We summarize the current findings regarding the role of TRP proteins in the development of cardiovascular disease, diabetes mellitus as well as diabetic complications, and tumorigenesis and present TRP proteins as targets of potential diagnostic and therapeutic strategies.
Assuntos
Canais de Potencial de Receptor Transitório/fisiologia , Animais , Carcinogênese/genética , Doenças Cardiovasculares/genética , Fenômenos Fisiológicos Cardiovasculares/genética , Canalopatias/complicações , Canalopatias/genética , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Humanos , Canais de Potencial de Receptor Transitório/genéticaRESUMO
TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Membrana/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Canais de Cátion TRPM/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Masculino , Potenciais da Membrana , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Dados de Sequência Molecular , Invasividade Neoplásica , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Canais de Cátion TRPM/genética , TransfecçãoRESUMO
Calcium channels significantly contribute to many cellular processes, including such critical ones as proliferation, differentiation, and apoptosis. In prostate cancer, the influence of calcium channels has been known for over 30 years, with the first observations that calcium channel blockers affect the progression of cancer towards more aggressive phase. Later research identified additional classes of channel proteins having an important regulatory role and affecting malignant transformation. This review discusses the accumulated scientific knowledge focused on calcium channel involvement in regulation of cell fate in prostate tissues as well as recent developments in patents targeted at prostate cancer treatments and involving calcium channel modulators.
Assuntos
Canais de Cálcio/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Humanos , Legislação de Medicamentos , Masculino , Patentes como Assunto , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologiaRESUMO
Ion channels contribute to virtually all basic cellular processes, including such crucial ones for maintaining tissue homeostasis as proliferation, differentiation, and apoptosis. The involvement of ion channels in regulation of programmed cell death, or apoptosis, has been known for at least three decades based on observation that classical blockers of ion channels can influence cell death rates, prolonging or shortening cell survival. Identification of the central role of these channels in regulation of cell cycle and apoptosis as well as the recent discovery that the expression of ion channels is not limited solely to the plasma membrane, but may also include membranes of internal compartments, has led researchers to appreciate the pivotal role of ion channels plays in development of cancer. This review focuses on the aspects of programmed cell death influenced by various ion channels and how dysfunctions and misregulations of these channels may affect the development and progression of different cancers.
Assuntos
Apoptose/fisiologia , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Animais , Humanos , Transporte de Íons/fisiologiaRESUMO
TRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca(2+) homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca(2+)-sensing elements of specific regulatory pathways leading to either proliferation or apoptosis is what makes these channels participate in cell fate regulation and, in turn, determines the final effect of Ca(2+) entry via the particular channel. This review focuses on the aspects of TRP channel localization and function that affect the balance between cell survival and death and how various dysregulations of these channels may lead to perturbed balance and onset of cancer.
Assuntos
Cálcio/metabolismo , Transformação Celular Neoplásica/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Morte Celular/fisiologia , Sobrevivência Celular , Humanos , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed.
Assuntos
Escherichia coli/fisiologia , Canais Iônicos/síntese química , Mycobacterium tuberculosis/fisiologia , Espectroscopia de Ressonância de Spin Eletrônica , Canais Iônicos/fisiologiaRESUMO
The mechanosensitive channel of large conductance from Mycobacterium tuberculosis (Tb-MscL) was subjected to cysteine-scanning mutagenesis at several residues in the M1 region. The V15C channel displayed disulfide crosslinking in air, but not in the presence of 100 mM beta-mercaptoethanol. In single-channel experiments, the V15C channel was more sensitive to tension than was wild-type Tb-MscL. In air, Tb-MscL V15C occasionally displayed signature-events: at constant tension, there was first a sojourn in the highest conductance open state, then a series of transitions to substates. During a signature-event, these transitions do not appear to be reversible. Some sojourns in the lower conductance states lasted for > or =100 s. These signature-events were abolished by 100 mM beta-mercaptoethanol and did not occur in a cysteineless gain-of-function mutant, suggesting that the signature-events represent disulfide crosslinking between channel subunits. We conclude that the crosslinking occurs during an open state during asymmetric sojourns that bring the alpha-carbons of adjacent 15C side chains within 3.6-6.8 A. Such asymmetric structures must be considered in models of TB-MscL gating.