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1.
Proc Natl Acad Sci U S A ; 115(35): E8201-E8210, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30108148

RESUMO

The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg2+ and Zn2+ Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg2+ ions occupying the center of the conduction pore. In high [Mg2+], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.


Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Evolução Molecular , Canais de Cátion TRPM/química , Animais , Camundongos , Domínios Proteicos , Canais de Cátion TRPM/metabolismo
2.
Elife ; 72018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30004384

RESUMO

We report the near atomic resolution (3.3 Å) of the human polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1.


Assuntos
Canais de Cálcio/ultraestrutura , Microscopia Crioeletrônica , Receptores de Superfície Celular/ultraestrutura , Cálcio , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Cátions/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo
3.
Chemistry ; 21(37): 12971-7, 2015 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-26315337

RESUMO

Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site-specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP-based approaches. We have examined in detail the effect of directly attaching mono- or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements. Using a membrane-embedded ion channel as an example, we varied the degree of paramagnetic labeling and the location of the DNP probes. Our results show that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of paramagnetic relaxation enhancement (PRE)-based NMR approaches. DNP using chemical labeling may hence provide an attractive route to introduce molecular specificity into DNP studies in life science applications and beyond.


Assuntos
Proteínas de Membrana/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica , Microscopia de Polarização , Ressonância Magnética Nuclear Biomolecular
4.
Biochemistry ; 53(16): 2557-9, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24621378

RESUMO

The potassium channel KcsA offers a unique opportunity to explicitly study the dynamics of the moving parts of ion channels, yet our understanding of the extent and dynamic behavior of the physiologically relevant structural changes at the inner gate in KcsA remains incomplete. Here, we use electron paramagnetic resonance, nuclear magnetic resonance, and molecular dynamics simulations to characterize the extent of pH-dependent conformational changes of the inner gate in lipid bilayers or detergent micelles. Our results show that under physiological conditions the inner gate experiences a maximal diagonal opening of ∼24 Šwith the largest degree of dynamics near the pKa of activation (pH ∼3.9). These results extend the observation that the C-terminus is necessary to limit the extent of opening and imply that the inner gate regulates the extent of conformational change at the zone of allosteric coupling and at the selectivity filter.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica
5.
Nat Struct Mol Biol ; 21(3): 244-52, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24487958

RESUMO

The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.


Assuntos
Ciona intestinalis/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Eletrofisiologia , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Xenopus laevis/metabolismo
6.
Proc Natl Acad Sci U S A ; 110(32): 13008-13, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23882077

RESUMO

Potassium (i.e., K(+)) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K(+) channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K(+) channel inactivation and exhibits a remarkable structural plasticity that correlates to K(+) channel inactivation. The transmembrane helix 1 unwinds when the K(+) channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K(+) ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K(+) channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K(+) channel pore.


Assuntos
Ativação do Canal Iônico/fisiologia , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Canais de Potássio/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Feminino , Ativação do Canal Iônico/genética , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus
7.
J Am Chem Soc ; 134(39): 16360-9, 2012 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-22946877

RESUMO

Distance determination from an echo intensity modulation obtained by pulsed double electron-electron resonance (DEER) experiment is a mathematically ill-posed problem. Tikhonov regularization yields distance distributions that can be difficult to interpret, especially in a system with multiple discrete distance distributions. Here, we show that by using geometric fit constraints in symmetric homo-oligomeric protein systems, we were able to increase the accuracy of a model-based fit solution based on a sum of Rice distributions. Our approach was validated on two different ion channels of known oligomeric states, KcsA (tetramer) and CorA (pentamer). Statistical analysis of the resulting fits was integrated within our method to help the experimenter evaluate the significance of a symmetry-constrained vs standard model distribution fit and to examine multidistance confidence regions. This approach was used to quantitatively evaluate the role of the C-terminal domain (CTD) on the flexibility and conformation of the activation gate of the K(+) channel KcsA. Our analysis reveals a significant increase in the dynamics of the inner bundle gate upon opening. Also, it explicitly demonstrates the degree to which the CTD restricts the motion of the lower gate at rest and during activation gating.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ativação do Canal Iônico , Canais de Potássio/química , Canais de Potássio/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Streptomyces lividans
8.
Cell ; 142(4): 515-6, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20723752

RESUMO

The mechanism by which voltage-dependent ion channels sense membrane potentials has been the most intensively studied and debated topic in modern ion channel research. In this issue, Xu et al. (2010) provide new insights into the minimal topological and physicochemical features required for voltage sensing.

9.
Vitam Horm ; 80: 635-48, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19251053

RESUMO

The short half-life of insulin in the human body (4-6 min) prompted the search and discovery of insulin-degrading enzyme (IDE), a 110-kDa metalloprotease that can rapidly degrade insulin into inactive fragments. Genetic and biochemical evidence accumulated in the last sixty years has implicated IDE as an important physiological contributor in the maintenance of insulin levels. Recent structural and biochemical analyses reveal the molecular basis of how IDE uses size and charge distribution of the catalytic chamber and structural flexibility of substrates to selectively recognize and degrade insulin, as well as the regulatory mechanisms of this enzyme. These studies provide a path for potential therapeutics in the control of insulin metabolism by the degradation of insulin.


Assuntos
Insulisina/química , Insulisina/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica , Humanos , Insulina/metabolismo , Insulisina/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Ratos
10.
J Neurosci ; 28(47): 12199-211, 2008 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19020014

RESUMO

In brain, monomeric immunoglobin G (IgG) is regarded as quiescent and only poised to initiate potentially injurious inflammatory reactions via immune complex formation associated with phagocytosis and tumor necrosis factor alpha (TNF-alpha) production in response to disease. Using rat hippocampal slice and microglial cultures, here we show instead that physiological levels (i.e., 0.2-20 microg/ml) of monomeric IgG unassociated with disease triggered benign low-level proinflammatory signaling that was neuroprotective against CA1 area excitotoxicity and followed a U-shaped or hormetic dose-response. The data indicate that physiological IgG levels activated microglia by enhancing recycling endocytosis plus TNF-alpha release from these cells to produce the neuroprotection. Minocycline, known for its anti-inflammatory and neuroprotective effects when given after disease onset, abrogated IgG-mediated neuroprotection and related microglial effects when given before injury. In contrast, E-prostanoid receptor subtype 2 (EP2) activation, which served as an exemplary paracrine stimulus like the one expected from neuronal activity, amplified IgG-mediated increased microglial recycling endocytosis and TNF-alpha production. Furthermore, like monomeric IgG these EP2 related effects took days to be effective, suggesting both were adaptive anabolic effects consistent with those seen from other long-term preconditioning stimuli requiring de novo protein synthesis. The data provide the first evidence that brain monomeric IgG at physiological levels can have signaling function via enhanced recycling endocytosis/TNF-alpha production from microglia unassociated with disease and that these IgG-mediated changes may be a means by which paracrine signaling from neuronal activity influences microglia to evoke neuroprotection. The data provide further support that low-level proinflammatory neural immune signaling unassociated with disease enhances brain function.


Assuntos
Endocitose/fisiologia , Imunoglobulina G/farmacologia , Microglia/efeitos dos fármacos , Microglia/fisiologia , Fármacos Neuroprotetores/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Ectodisplasinas/metabolismo , Endocitose/efeitos dos fármacos , Glucose/deficiência , Hipocampo , Hipóxia , Imunoglobulina G/líquido cefalorraquidiano , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Minociclina/farmacologia , N-Metilaspartato/toxicidade , Neurotoxinas/toxicidade , Técnicas de Cultura de Órgãos , Fosfopiruvato Hidratase/metabolismo , Ratos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rab de Ligação ao GTP/metabolismo
11.
J Neurosci ; 25(15): 3952-61, 2005 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-15829647

RESUMO

Spreading depression (SD) involves current flow through principal neurons, but the pattern of current flow over the expanse of susceptible tissues or individual principal neurons remains undefined. Accordingly, tissue and single cell maps made from digital imaging of voltage-sensitive dye changes in hippocampal organotypic cultures undergoing SD were processed via optical current source density analysis to reveal the currents associated with pyramidal neurons. Two distinctive current flow patterns were seen. The first was a trilaminar pattern (420 microm2) that developed with the onset of SD in CA3 pyramidal neurons, in which SD most often began. This initial pattern comprised a somatic current sink with current sources to either side in the dendrites that lasted for seconds extending into the first aspect of the classical "inverted saddle" interstitial direct current waveform of SD. Next, the somatic sink backpropagated at a speed of millimeters per minute into the proximal dendrites, resulting in a reversal of the initial current flow pattern to its second orientation, namely dendritic sinks associated with a somatic source. The latter persisted for the remainder of SD in CA3 and was the only pattern seen in CA1, in which SD was rarely initiated. This backpropagating SD current flow resembles that of activity-dependent synaptic activation. Retrograde and associative signaling via principal neuron current flow is a key means to affect tissue function, including synaptic activation and, by extension, perhaps SD. Such current-related postsynaptic signaling might not only help explain SD but also neuroprotection and migraine, two phenomena increasingly recognized as being related to SD.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Hipocampo/citologia , Células Piramidais/fisiologia , Animais , Animais Recém-Nascidos , Diagnóstico por Imagem/métodos , Estimulação Elétrica/métodos , Imuno-Histoquímica/métodos , Lisina/análogos & derivados , Lisina/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Modelos Neurológicos , Proteínas de Neurofilamentos/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Wistar , Estatísticas não Paramétricas , Estirenos/metabolismo , Fatores de Tempo
12.
J Cereb Blood Flow Metab ; 24(8): 829-39, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15362713

RESUMO

Cytokines are involved in ischemic tolerance, including that triggered by spreading depression (SD), yet their roles in neuroprotection remain incompletely defined. The latter may stem from the pleiotropic nature of these signaling molecules whose complexities for interaction might be better deciphered through simultaneous measurement of multiple targeted proteins. Accordingly, the authors used microsphere-based flow cytometric immunoassays and hippocampal organotypic cultures (HOTCs) to characterize the magnitude, time course, and diversity of cytokine (interleukin [IL] 1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) response to SD. GM-CSF was not detected in HOTCs or media. However, SD triggered a significant, generalized increase in seven cytokines evident in HOTCs 6 hours later, with the remaining cytokine, IL-1beta, becoming significantly different at 1 and 3 days. Additionally, these changes extended to include surrounding media for IL-6 and TNF-alpha by 1 and 3 days. This increase was localized to microglia via immunostaining for IL-1alpha, IL-1beta, and interferon-y. IL-10, although significantly more abundant in HOTCs 6 hours after SD, was significantly less abundant in surrounding media at that time and at 1 day. Finally, the generalized early increase in tissue cytokines later settled to a pattern at 3 days of recovery centering on changes in IL-1alpha, IL-1beta, and TNF-alpha, cytokines capable of modulating ischemic injury.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Citocinas/biossíntese , Hipocampo/fisiologia , Animais , Estimulação Elétrica , Eletrofisiologia , Citometria de Fluxo , Imunoensaio , Imuno-Histoquímica , Microesferas , Técnicas de Cultura de Órgãos
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