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2.
Toxicol Appl Pharmacol ; 449: 116073, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35605787

RESUMO

The anthelmintic ivermectin has been reported to possess anticancer and antiviral efficacy. However, the effective concentrations reported in vitro are near the predicted aqueous solubility limit for this hydrophobic drug. We observed that ivermectin-induced cell death in two cervical cancer cell lines correlated with the formation of solid ivermectin aggregates in both serum-free and serum-supplemented culture media. Filtration of ivermectin particles >0.2 µm abolished these cytolytic effects in both cell lines. An inhibitory effect on cell proliferation persisted for filtered solutions, but only for ivermectin concentrations higher than reported to be clinically attainable in humans. In addition to the importance of distinguishing between free and bound drug in solution, our data emphasize the importance of acknowledging the likely solubility limit of hydrophobic drugs when assessing their in vitro cytotoxicity.


Assuntos
Anti-Helmínticos , Neoplasias do Colo do Útero , Morte Celular , Meios de Cultura , Feminino , Humanos , Ivermectina/toxicidade
3.
J Biol Chem ; 296: 100084, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33199365

RESUMO

The poor and nonselective penetration of current chemotherapeutics across the plasma membranes of cancer cells, which is necessary for the targeted disruption of the intracellular machinery, remains a major pharmaceutical challenge. In several cell types, including mast cells and macrophages, exposure to extracellular ATP is known to stimulate passive entry of large and otherwise membrane impermeable cationic dyes, which is usually attributed to conduction through ionotropic P2X receptors. Here, we report that elevations in cytosolic Ca2+ stimulate the rapid uptake and nuclear accumulation of a DNA-binding fluorescent cation, Hoechst 33258 (H33258), in cervical cancer cells. The H33258 uptake was dependent on activation of intermediate conductance Ca2+-activated K+ channels (KCa3.1), and direct stimulation of the channel with the activators SKA 31 and DCEBIO was sufficient to induce cellular uptake of H33258 directly. In contrast to the results from cancerous cervical cells, KCa3.1-dependent H33258 uptake was rarely observed in epithelial cells derived from the ectocervix and transformation zone of healthy cervical tissue. Furthermore, whole-cell patch clamp experiments and assessment of membrane potential using the slow voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid)trimethine oxonol revealed a significant difference in functional KCa3.1 activity between cancerous and healthy cervical epithelial cells, which correlated strongly with the incidence of KCa3.1-dependent H33258 uptake. Finally, we show that activation of KCa3.1 channels caused a modest but significant sensitization of cancer cells to the growth suppressant effects of H33258, lending plausibility to the idea of using KCa3.1 channel activators to enhance cell penetration of small cationic toxins into cancer cells expressing these channels.


Assuntos
Benzimidazóis , Citotoxinas , DNA de Neoplasias/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias do Colo do Útero/metabolismo , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Transporte Biológico Ativo , Linhagem Celular Tumoral , Citotoxinas/farmacocinética , Citotoxinas/farmacologia , Feminino , Humanos , Neoplasias do Colo do Útero/patologia
4.
Macromol Biosci ; 20(5): e1900377, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32207234

RESUMO

The correlation between erosion and drug (lidocaine and 6-mercaptopurine, 6-MP) release from amorphous poly(thioether anhydrides), which are synthesized using radical-mediated thiol-ene polymerization, is reported. Cytotoxicity studies of the polymer toward human fibroblast human dermal fibroblasts adult, melanoma A-375, and breast cancer MCF-7 cells are conducted, and drug efficacy of a cancer and autoimmune disease drug (6-MP) when released from the poly(thioether anhydrides) is examined against two cancerous cell types (A-375 and MCF-7). Erosion and drug release studies reveal that lidocaine release is governed by network erosion whereas 6-MP is released by a combination of erosion and diffusion. The cytotoxicity studies show that all three cell types demonstrate high viability, thus cytocompatibility, to poly(thioether anhydrides). Toxicity to the material is dose dependent and comparable to other polyanhydride systems. The 6-MP cancer drug is shown to remain bioactive after encapsulation in the poly(thioether anhydride) matrix and the polymer does not appear to modify the efficacy of the drug.


Assuntos
Anidridos/química , Sistemas de Liberação de Medicamentos , Sulfetos/química , Adulto , Anidridos/síntese química , Contagem de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Cinética , Lidocaína/farmacologia , Mercaptopurina/farmacologia , Sulfetos/síntese química
5.
Adv Exp Med Biol ; 1140: 359-375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347058

RESUMO

Ion channels are intrinsic membrane proteins that form gated ion permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca2+, modulating a multitude of cell signaling systems in the process. The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca2+ levels, and the fact that they are particularly amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure relates to their function. Historically, elucidation of the structure of membrane proteins has been slow relative to that for soluble proteins, due to limitations inherent in the most widely used methods, in particular X-ray crystallography. Over the course of the last decade, starting with significant advances in X-ray crystallography followed by the more recent, and profound, surge in the use of single particle cryo-electron microscopy (cryo-EM), a slew of high resolution ion channel structures have been resolved. Overshadowed during this period have been the equally marked advances in mass spectrometry, pushing this method to the fore as an important complimentary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.


Assuntos
Canais Iônicos/fisiologia , Espectrometria de Massas , Transdução de Sinais , Membrana Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteômica
6.
J Biol Chem ; 294(33): 12521-12533, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31248985

RESUMO

Activation of ionotropic P2X receptors increases free intracellular Ca2+ ([Ca2+] i ) by initiating a transmembrane cation flux. We studied the "a" and "k" splice variants of the rat purinergic P2X7 receptor (rP2X7aR and rP2X7kR) to exhibit a significant difference in Ca2+ flux through this channel. This difference is surprising because the variants share absolute sequence identity in the area of the pore that defines ionic selectivity. Here, we used patch-clamp fluorometry and chimeric receptors to show that the fraction of the total current carried by Ca2+ is a function of the primary sequence of the cytoplasmic N terminus. Using scanning mutagenesis, we identified five sites within the N terminus that respond to mutagenesis with a decrease in fractional calcium current and an increase in permeability to the polyatomic cation, N-methyl-d-glucamine (NMDG+), relative to Na+ (PNMDG/PNa). We tested the hypothesis that these sites line the permeation pathway by measuring the ability of thiol-reactive MTSET+ to alter the current of cysteine-substituted variants, but we detected no effect. Finally, we studied the homologous sites of the rat P2X2 receptor (rP2X2R) and observed that substitutions at Glu17 significantly reduced the fractional calcium current. Taken together, our results suggest that a change in the structure of the N terminus alters the ability of an intra-pore Ca2+ selectivity filter to discriminate among permeating cations. These results are noteworthy for two reasons: they identify a previously unknown outcome of mutagenesis of the N-terminal domain, and they suggest caution when assigning structure to function for truncated P2X receptors that lack a part of the N terminus.


Assuntos
Processamento Alternativo , Sinalização do Cálcio , Cálcio/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Substituição de Aminoácidos , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Purinérgicos P2X7/genética
7.
Bioact Mater ; 2(4): 260-268, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29744435

RESUMO

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy. In this contribution, we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides (MDPs) and use them as a model system to investigate structure-dependent cell penetrating activity. MDPs self-assembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane. However, despite the multivalent charge presentation, not all fibers led to high levels of membrane activity and cellular uptake. The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation. Nanofibers were found to sacrifice their dimension, thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction. The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation. In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone. Through these detailed structure and activity studies, the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.

8.
Cell Biol Toxicol ; 32(5): 363-71, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27423453

RESUMO

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.


Assuntos
Corantes Fluorescentes/farmacocinética , Canais Iônicos/metabolismo , Compostos de Quinolínio/farmacocinética , Trifosfato de Adenosina/metabolismo , Benzoxazóis/farmacocinética , Cátions/farmacocinética , Sobrevivência Celular/fisiologia , Células HEK293 , Humanos , Indóis/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Canais de Cátion TRPV/metabolismo
9.
Mol Biosyst ; 12(9): 2695-9, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27397555

RESUMO

Self-assembly of de novo designed multidomain peptides (MDPs) resulted in functional membrane-active supramolecular nanofibers. The membrane activity was analyzed through fluorescence membrane localization and patch-clamp electrophysiology yielding important information that can be used for the development of a new type of supramolecular peptide-based chemotherapeutic enhancer.


Assuntos
Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Membrana Celular/química , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Sinergismo Farmacológico , Humanos , Metabolismo dos Lipídeos , Lipídeos/química , Potenciais da Membrana/efeitos dos fármacos , Peptídeos/metabolismo
10.
J Biomed Mater Res A ; 104(8): 1936-45, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27012532

RESUMO

Quantitative and qualitative toxicological analyses of crosslinked, surface-eroding polyanhydrides (PAHs) made from thiol-ene "click" polymerizations are reported. The cytotoxicity of these PAHs was investigated against three skin-based cell types; melanoma (A-375), human dermal fibroblast adult (HDFa), and 3T3-J2 (mouse fibroblast) cells, thus providing insight into the potential for these PAHs to be used in dermal drug delivery applications. Apoptosis was evaluated quantitatively and qualitatively using MTT assay and fluorescence microscopic imaging as indication of cytotoxicity. Upon exposure of A-375 and HDFa cells to high concentrations (4000 mg/L) of crosslinked PAH, the respective morphologies remained relatively unchanged compared with nonexposed cells. The 3T3-J2 cell type was more sensitive towards the PAH, exhibiting minimal deformation of cell morphology at 4000 mg/L. The MTT assay and fluorescence imaging revealed that this PAH and its degradation products are highly cytocompatible at high concentrations and cytotoxicity observed is dosage/time dependent. Further, the PAH did not induce inhibition of tested cells' proliferation at high polymer concentration up to 2000 mg/L. The IC50 (concentration of the crosslinked PAH required to inhibit 50% cell viability) for HDFa and A-375 cells was determined to be 4300 ± 70 and 8500 ± 50 mg/L, respectively. The high cytocompatibility of this type of crosslinked PAH, in addition to their degradation products, towards these skin cells (standard and cancer cell types) suggests that the polymer may be viable for dermal-based drug delivery to normal and cancerous diseased tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1936-1945, 2016.


Assuntos
Química Click/métodos , Polianidridos/toxicidade , Compostos de Sulfidrila/química , Testes de Toxicidade , Células 3T3 , Adulto , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Derme/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Polianidridos/química , Polimerização
11.
Pflugers Arch ; 468(2): 201-11, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26490461

RESUMO

Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.


Assuntos
Cálcio/metabolismo , Canais de Cátion TRPV/metabolismo , Potenciais de Ação , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Permeabilidade
13.
FEBS Lett ; 589(13): 1498-504, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25937122

RESUMO

Extracellular ATP is known to permeabilize certain cell types to polyatomic cations like YO-PRO1. Here, we report that extracellularly applied ATP stimulated rapid uptake and accumulation of an otherwise weakly membrane permeable fluorescent DNA-binding cytotoxin, Hoechst 33258, into cervical cancer cells. While ATP stimulated Hoechst 33258 uptake in 20-70% of cells from seven cervical cancer cell lines, it stimulated uptake in less than 8% of cervical epithelial cells obtained from the normal transformation zone and ectocervix tissue of 11 patients. ATP-evoked Hoechst 33258 uptake was independent of ionotropic P2X receptors, but dependent on activation of P2Y receptors. Thus, we show here that cervical cancer cells can be selectively induced to take up and accumulate an ionic cytotoxin by exposure to extracellular ATP.


Assuntos
Benzimidazóis/farmacocinética , Permeabilidade da Membrana Celular , Citotoxinas/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Benzimidazóis/química , Benzimidazóis/metabolismo , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Citotoxinas/química , Citotoxinas/farmacocinética , DNA de Neoplasias/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Uridina Trifosfato/farmacologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
14.
Purinergic Signal ; 11(2): 229-38, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25847072

RESUMO

P2X receptors are cation-permeable ion channels gated by extracellular adenosine triphosphate (ATP). Available crystallographic data suggest that ATP-binding ectodomain is connected to the transmembrane pore domain by three structurally conserved linker regions, which additionally frame the lateral fenestrations through which permeating ions enter the channel pore. The role of these linker regions in relaying the conformational change evoked by ATP binding of the ectodomain to the pore-forming transmembrane domain has not been investigated systematically. Using P2X4R as our model, we employed alanine and serine replacement mutagenesis to determine how the side chain structure of these linker regions influences gating. The mutants Y54A/S, F198A/S, and W259A/S all trafficked normally to the plasma membrane of transfected HEK293 cells but were poorly responsive to ATP. Nevertheless, the function of the F198A/S mutants could be recovered by pretreatment with the known positive allosteric modulator of P2X4R, ivermectin (IVM), although the IVM sensitivity of this mutant was significantly impaired relative to wild type. The functional mutants Y195A/S, F200A/S, and F330A/S exhibited ATP sensitivities identical to wild type, consistent with these side chains playing no role in ATP binding. However, Y195A/S, F200A/S, and F330A/S all displayed markedly changed sensitivity to the specific effects of IVM on current deactivation, suggesting that these positions influence allosteric modulation of gating. Taken together, our data indicate that conserved amino acids within the regions linking the ectodomain with the pore-forming transmembrane domain meaningfully contribute to signal transduction and channel gating in P2X receptors.


Assuntos
Antiparasitários/farmacologia , Ivermectina/farmacologia , Receptores Purinérgicos P2X4/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida/métodos , Técnicas de Patch-Clamp/métodos , Estrutura Terciária de Proteína/genética , Ratos , Transdução de Sinais/efeitos dos fármacos
15.
J Biol Chem ; 290(12): 7930-42, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25645917

RESUMO

ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca(2+) through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca(2+) is universally acknowledged, the biophysics of the Ca(2+) flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca(2+) permeability are difficult to apply to P2X7 receptors. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca(2+) flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca(2+) component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca(2+) to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca(2+) current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca(2+) through the channel.


Assuntos
Trifosfato de Adenosina/fisiologia , Canais de Cálcio/metabolismo , Receptores Purinérgicos P2X7/fisiologia , Animais , Células Cultivadas , Humanos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Camundongos , Permeabilidade
16.
Adv Exp Med Biol ; 806: 237-61, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24952185

RESUMO

Ion channels are intrinsic membrane proteins that form gated ion-permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca(2+). The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca(2+) levels, and the fact that they are most amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure imparts function. Here, progress has been slow relative to that for soluble protein structures in large part due to the limitations of applying conventional structure determination methods, such as X-ray crystallography, nuclear magnetic resonance imaging, and mass spectrometry, to membrane proteins. Although still an underutilized technique in the assessment of membrane protein structure, recent advances have pushed mass spectrometry to the fore as an important complementary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high-throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular-signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.


Assuntos
Canais Iônicos , Espectrometria de Massas/métodos , Complexos Multiproteicos , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Processamento Alternativo/fisiologia , Animais , Cristalografia por Raios X , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Relação Estrutura-Atividade
17.
Biomacromolecules ; 15(7): 2573-82, 2014 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-24848134

RESUMO

Several critical aspects of cross-linked polyanhydrides made using thiol-ene polymerization are reported, in particular the erosion, release, and solution properties, along with their cytotoxicity toward fibroblast cells. The monomers used to synthesize these polyanhydrides were 4-pentenoic anhydride and pentaerythritol tetrakis(3-mercaptopropionate). Techniques used to evaluate the erosion mechanism indicate a complex situation in which several phenomena, such as hydrolysis rates, local pH, water diffusion, and solubility, may be influencing the erosion process. The mass loss profile, the release rate of a hydrophilic dye, the rate of hydrolysis of the polyanhydride, the hydrolysis product solubility as a function of pH, average pK(a) and its cytotoxicity toward fibroblast cells were all determined. The solubility of the degradation product is low at pH values less than 6-7, and the average pKa was determined to be ~5.3. The cytotoxicity of the polymer and the degradation product was found to be low, with cell viabilities of >97% for the various samples studied at concentrations of ~1000-1500 ppm. These important parameters help determine the potential of the thiol-ene polyanhydrides in various biomedical applications. These polyanhydrides can be used as a delivery vehicle, and although the release profile qualitatively followed the mass loss profile for a hydrophilic dye, the release rate appears to be by both diffusion and mass loss mechanisms.


Assuntos
Polianidridos/síntese química , Materiais Biocompatíveis/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos/química , Humanos , Hidrólise , Cinética , Processos Fotoquímicos , Polianidridos/toxicidade , Polimerização , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/toxicidade
18.
Biochemistry ; 53(18): 3012-9, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24762105

RESUMO

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.


Assuntos
Trifosfato de Adenosina/metabolismo , Leucina/química , Receptores Purinérgicos P2X4/química , Substituição de Aminoácidos , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Ivermectina/farmacologia , Leucina/genética , Leucina/metabolismo , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Ratos , Receptores Purinérgicos P2X4/efeitos dos fármacos , Transdução de Sinais
19.
Front Cell Neurosci ; 8: 6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24550775

RESUMO

P2X receptors are a family of trimeric ion channels that are gated by extracellular adenosine 5'-triphosphate (ATP). These receptors have long been a subject of intense research interest by virtue of their vital role in mediating the rapid and direct effects of extracellular ATP on membrane potential and cytosolic Ca(2+) concentration, which in turn underpin the ability of ATP to regulate a diverse range of clinically significant physiological functions, including those associated with the cardiovascular, sensory, and immune systems. An important aspect of an ion channel's function is, of course, the means by which it transports ions across the biological membrane. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of how P2X receptors conduct the inward flux of Na(+) and Ca(2+) in response to binding by ATP. However, this work has relied heavily on results from current recordings of P2X receptors altered by site-directed mutagenesis. In the absence of a 3-dimensional channel structure, this prior work provided only a vague and indirect appreciation of the relationship between structure, ion selectivity and flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor has thus proved a significant boon, and has provided an important opportunity to overview the amassed functional data in the context of a working 3-dimensional model of a P2X receptor. In this paper, we will attempt to reconcile the existing functional data regarding ion permeation through P2X receptors with the available crystal structure data, highlighting areas of concordance and discordance as appropriate.

20.
J Biol Chem ; 287(10): 7594-602, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22219189

RESUMO

Human P2X receptors are a family of seven ATP-gated ion channels that transport Na(+), K(+), and Ca(2+) across cell surface membranes. The P2X4 receptor is unique among family members in its sensitivity to the macrocyclic lactone, ivermectin, which allosterically modulates both ion conduction and channel gating. In this paper we show that removing the fixed negative charge of a single acidic amino acid (Glu(51)) in the lateral entrance to the transmembrane pore markedly attenuates the effect of ivermectin on Ca(2+) current and channel gating. Ca(2+) entry through P2X4 receptors is known to trigger downstream signaling pathways in microglia. Our experiments show that the lateral portals could present a novel target for drugs in the treatment of microglia-associated disease including neuropathic pain.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Substituição de Aminoácidos , Antiparasitários/farmacologia , Linhagem Celular Transformada , Humanos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Ivermectina/farmacologia , Microglia/patologia , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Neuralgia/patologia , Receptores Purinérgicos P2X4/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
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