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1.
Nature ; 580(7802): 263-268, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269334

RESUMO

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.


Assuntos
Canais de Cloreto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ingestão de Alimentos/fisiologia , Intestinos/fisiologia , Zinco/metabolismo , Animais , Drosophila melanogaster/genética , Enterócitos/metabolismo , Feminino , Preferências Alimentares , Homeostase , Insetos Vetores , Insulina/metabolismo , Ativação do Canal Iônico , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lisossomos/metabolismo , Masculino , Oócitos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Xenopus
2.
PLoS Comput Biol ; 16(3): e1007530, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226009

RESUMO

This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.


Assuntos
Canais de Cloreto/genética , Ativação do Canal Iônico/fisiologia , Animais , Bovinos , Cloretos/metabolismo , Biologia Computacional/métodos , Cinética , Cadeias de Markov , Potenciais da Membrana , Modelos Biológicos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação
3.
Nature ; 580(7802): 288-293, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269335

RESUMO

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.


Assuntos
Microscopia Crioeletrônica , Ativação do Canal Iônico , Methanobacterium/química , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/ultraestrutura , Cálcio/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/metabolismo , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Termodinâmica
4.
Phys Rev Lett ; 124(4): 048102, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32058787

RESUMO

Experiments have suggested that bacterial mechanosensitive channels separate into 2D clusters, the role of which is unclear. By developing a coarse-grained computer model we find that clustering promotes the channel closure, which is highly dependent on the channel concentration and membrane stress. This behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We implement this positive feedback into the model for cell volume regulation, and find that the channel clustering protects the cell against excessive loss of cytoplasmic content.


Assuntos
Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Biológicos , Modelos Químicos , Escherichia coli/citologia , Escherichia coli/metabolismo , Ativação do Canal Iônico , Mecanotransdução Celular
5.
Nat Commun ; 11(1): 1039, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098964

RESUMO

Genetically encoded photoswitches have enabled spatial and temporal control of cellular events to achieve tailored functions in living cells, but their applications to probe the structure-function relations of signaling proteins are still underexplored. We illustrate herein the incorporation of various blue light-responsive photoreceptors into modular domains of the stromal interaction molecule 1 (STIM1) to manipulate protein activity and faithfully recapitulate STIM1-mediated signaling events. Capitalizing on these optogenetic tools, we identify the molecular determinants required to mediate protein oligomerization, intramolecular conformational switch, and protein-target interactions. In parallel, we have applied these synthetic devices to enable light-inducible gating of calcium channels, conformational switch, dynamic protein-microtubule interactions and assembly of membrane contact sites in a reversible manner. Our optogenetic engineering approach can be broadly applied to aid the mechanistic dissection of cell signaling, as well as non-invasive interrogation of physiological processes with high precision.


Assuntos
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Optogenética/métodos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Cálcio/metabolismo , Criptocromos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ativação do Canal Iônico , Proteínas Luminescentes/genética , Mutação , Proteínas de Neoplasias/química , Neoplasias/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Molécula 1 de Interação Estromal/química , Relação Estrutura-Atividade
6.
Nat Commun ; 11(1): 676, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015334

RESUMO

In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Here, utilizing KV7.1's unique two open states, we report a two-stage E-M coupling mechanism in voltage-dependent gating of KV7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore of the neighboring subunit to activate conductance. This two-stage hand-and-elbow gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous domain-swapped KV channels.


Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/fisiologia , Simulação de Acoplamento Molecular , Oócitos/metabolismo , Canais de Potássio , Conformação Proteica
7.
Life Sci ; 243: 117293, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31930971

RESUMO

Ca2+ overload in neurons has been implicated in Alzheimer's Disease (AD). Upregulation of Ca2+ through L-type Ca2+ channels was known to be involved in the neurodegeneration induced by amyloid-ß (Aß) peptides in AD. However, little is known about the mechanism by which upregulation of L-type Ca2+ channel currents is linked to Aß-induced neuronal toxicity. In the present study, we found that the L-type Ca2+ current in transgenic AD mice (Tg2576) neurons is greater than in wild-type (WT) neurons, and this Ca2+ channel current change were rescued in Tg2576/p75NTR+/- (p75 neurotrophin receptor) neurons. We further examined the changes in the gating of L-type Ca2+ channels following Aß42 treatment, and the results showed that the L-type Ca2+ channel current was significantly increased by Aß42 treatment in WT hippocampal neurons. Blocking or decreasing the expression of p75NTR eliminated the influence of Aß42 on the L-type Ca2+ channel current in WT hippocampal neurons. We also evaluated how Aß42 affected the voltage-dependent activation and inactivation of L-type Ca2+ channels in cultured WT neurons. The results indicated that the half-maximal activation voltage (V1/2) was left shifted, and the half-inactivation voltage (V1/2) displayed a right shift in neuron treated by Aß42. Decreasing the expression of p75NTR eliminated the effect of Aß42 on voltage-dependent activation and inactivation of the L-type Ca2+ channel. These results indicate that Aß42 changes L-type Ca2+ channel currents by modulating the channel's activation and inactivation dynamics, while decreasing p75NTR expression can remove this effect.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Canais de Cálcio Tipo L/metabolismo , Neurônios/metabolismo , Receptor de Fator de Crescimento Neural/fisiologia , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Humanos , Ativação do Canal Iônico , Camundongos , Camundongos Transgênicos
8.
Bioelectrochemistry ; 132: 107449, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31918058

RESUMO

Dysregulation of the cardiac ryanodine receptor (RYR2) by luminal Ca2+ has been implicated in a life-threatening, stress-induced arrhythmogenic disease. The mechanism of luminal Ca2+-mediated RYR2 regulation is under debate, and it has been attributed to Ca2+ binding on the cytosolic face (the Ca2+ feedthrough mechanism) and/or the luminal face of the RYR2 channel (the true luminal mechanism). The molecular nature and location of the luminal Ca2+ site is unclear. At the single-channel level, we directly probed the RYR2 luminal face by Eu3+, considering the non-permeant nature of trivalent cations and their high binding affinities for Ca2+ sites. Without affecting essential determinants of the Ca2+ feedthrough mechanism, we found that luminal Eu3+ competitively antagonized the activation effect of luminal Ca2+ on RYR2 responsiveness to cytosolic caffeine, and no appreciable effect was observed for luminal Ba2+ (mimicking the absence of luminal Ca2+). Importantly, luminal Eu3+ caused no changes in RYR2 gating. Our results indicate that two distinct Ca2+ sites (available for luminal Ca2+ even when the channel is closed) are likely involved in the true luminal mechanism. One site facing the lumen regulates channel responsiveness to caffeine, while the other site, presumably positioned in the channel pore, governs the gating behavior.


Assuntos
Cálcio/metabolismo , Európio/química , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cafeína/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos
9.
Int J Mol Sci ; 21(2)2020 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-31940951

RESUMO

Evidence that neighboring cells uncouple from each other as one dies surfaced in the late 19th century, but it took almost a century for scientists to start understanding the uncoupling mechanism (chemical gating). The role of cytosolic free calcium (Ca2+i) in cell-cell channel gating was first reported in the mid-sixties. In these studies, only micromolar [Ca2+]i were believed to affect gating-concentrations reachable only in cell death, which would discard Ca2+i as a fine modulator of cell coupling. More recently, however, numerous researchers, including us, have reported the effectiveness of nanomolar [Ca2+]i. Since connexins do not have high-affinity calcium sites, the effectiveness of nanomolar [Ca2+]i suggests the role of Ca-modulated proteins, with calmodulin (CaM) being most obvious. Indeed, in 1981 we first reported that a CaM-inhibitor prevents chemical gating. Since then, the CaM role in gating has been confirmed by studies that tested it with a variety of approaches such as treatments with CaM-inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent co-localization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as CaM targets. Our gating model envisions Ca2+-CaM to directly gate the channels by acting as a plug ("Cork" gating model), and probably also by affecting connexin conformation.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Junções Comunicantes/metabolismo , Animais , Calmodulina/genética , Citosol/metabolismo , Humanos , Ativação do Canal Iônico , Transdução de Sinais
10.
Nat Chem Biol ; 16(2): 188-196, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959964

RESUMO

Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.


Assuntos
Pirrolidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glicina/metabolismo , Glicina/farmacologia , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevis
12.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R390-R398, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913684

RESUMO

Local skin heating to 42°C causes cutaneous thermal hyperemia largely via nitric oxide (NO) synthase (NOS)-related mechanisms. We assessed the hypothesis that ATP-sensitive K+ (KATP) channels interact with NOS to mediate cutaneous thermal hyperemia. In 13 young adults (6 women, 7 men), cutaneous vascular conductance (CVC) was measured at four intradermal microdialysis sites that were continuously perfused with 1) lactated Ringer solution (control), 2) 5 mM glibenclamide (KATP channel blocker), 3) 20 mM NG-nitro-l-arginine methyl ester (NOS inhibitor), or 4) a combination of KATP channel blocker and NOS inhibitor. Local skin heating to 42°C was administered at all four treatment sites to elicit cutaneous thermal hyperemia. Thirty minutes after the local heating, 1.25 mM pinacidil (KATP channel opener) and subsequently 25 mM sodium nitroprusside (NO donor) were administered to three of the four sites (each 25-30 min). The local heating-induced prolonged elevation in CVC was attenuated by glibenclamide (19%), but the transient initial peak was not. However, glibenclamide had no effect on the prolonged elevation in CVC in the presence of NOS inhibition. Pinacidil caused an elevation in CVC, but this response was abolished at the glibenclamide-treated skin site, demonstrating its effectiveness as a KATP channel blocker. The pinacidil-induced increase in CVC was unaffected by NOS inhibition, whereas the increase in CVC elicited by sodium nitroprusside was partly (15%) inhibited by glibenclamide. In summary, we showed an interactive effect of KATP channels and NOS for the plateau of cutaneous thermal hyperemia. This interplay may reflect a vascular smooth muscle cell KATP channel activation by NO.


Assuntos
Hiperemia/enzimologia , Canais KATP/metabolismo , Microcirculação , Microvasos/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Pele/irrigação sanguínea , Vasodilatação , Adulto , Velocidade do Fluxo Sanguíneo , Inibidores Enzimáticos/administração & dosagem , Feminino , Humanos , Hiperemia/etiologia , Hiperemia/fisiopatologia , Hipotermia Induzida , Ativação do Canal Iônico , Canais KATP/antagonistas & inibidores , Masculino , Microcirculação/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Microvasos/fisiopatologia , Doadores de Óxido Nítrico/administração & dosagem , Óxido Nítrico Sintase/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/administração & dosagem , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Adulto Jovem
13.
Proc Natl Acad Sci U S A ; 117(4): 1988-1993, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31924746

RESUMO

Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ∼ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration.


Assuntos
Ativação do Canal Iônico/fisiologia , Sódio/metabolismo , Potenciais de Ação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica
14.
Am J Physiol Cell Physiol ; 318(1): C150-C162, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31721612

RESUMO

Epithelial Na+ channels (ENaCs) are members of a family of cation channels that function as sensors of the extracellular environment. ENaCs are activated by specific proteases in the biosynthetic pathway and at the cell surface and remove embedded inhibitory tracts, which allows channels to transition to higher open-probability states. Resolved structures of ENaC and an acid-sensing ion channel revealed highly organized extracellular regions. Within the periphery of ENaC subunits are unique domains formed by antiparallel ß-strands containing the inhibitory tracts and protease cleavage sites. ENaCs are inhibited by Na+ binding to specific extracellular site(s), which promotes channel transition to a lower open-probability state. Specific inositol phospholipids and channel modification by Cys-palmitoylation enhance channel open probability. How these regulatory factors interact in a concerted manner to influence channel open probability is an important question that has not been resolved. These various factors are reviewed, and the impact of specific factors on human disorders is discussed.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/química , Humanos , Potenciais da Membrana , Modelos Moleculares , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
15.
Mol Pharmacol ; 97(2): 132-144, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31722973

RESUMO

The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (I Ks ), which is partly responsible for cardiomyocyte repolarization and physiologic shortening of the cardiac action potential. Mefenamic acid, a nonsteroidal anti-inflammatory drug, has been identified as an I Ks activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid's effect on I Ks Using whole-cell patch clamp, we show that mefenamic acid enhances I Ks activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating I Ks current into an almost linear current with instantaneous onset and slowed tail current decay, sensitive to the I Ks blocker (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide (HMR1556). Both single channels, which reveal no change in the maximum conductance, and whole-cell studies, which reveal a dramatically altered conductance-voltage relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the I Ks channel and shifts channel gating kinetics toward more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose and frequency dependence of mefenamic acid action on I Ks channels. Mutational analysis showed that mefenamic acid's effect on I Ks required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all I Ks channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT: The channels which generate the cardiac slow delayed rectifier K+ current (I Ks ) are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by I Ks in heartbeat regulation, enhancing I Ks current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most I Ks activators, unfortunately, only work on KCNQ1 alone and not the physiologically relevant I Ks channel. We have demonstrated that mefenamic acid can enhance I Ks in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.


Assuntos
Antiarrítmicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ácido Mefenâmico/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Animais , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Relação Dose-Resposta a Droga , Fibroblastos , Células HEK293 , Frequência Cardíaca/fisiologia , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Miocárdio/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/agonistas , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Cell Mol Life Sci ; 77(4): 765-778, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31327045

RESUMO

Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.


Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/química , Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas de Peixe-Zebra/química
17.
Biochim Biophys Acta Biomembr ; 1862(2): 183129, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738900

RESUMO

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.


Assuntos
Substituição de Aminoácidos , Cisteína/genética , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Cisteína/química , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/química , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Ligação Proteica , Domínios Proteicos
18.
Am J Physiol Lung Cell Mol Physiol ; 318(2): L366-L375, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31800260

RESUMO

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and ß-subunit (BKß1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.


Assuntos
Brônquios/citologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Subunidades Proteicas/metabolismo , Acetofenonas/farmacologia , Animais , Benzopiranos/farmacologia , Cálcio/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos Wistar
20.
Methods Mol Biol ; 2041: 243-259, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646494

RESUMO

Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.


Assuntos
Trifosfato de Adenosina/metabolismo , Biotinilação/métodos , Membrana Celular/metabolismo , Eletrofisiologia/métodos , Oócitos/fisiologia , Receptores Purinérgicos P2X/fisiologia , Xenopus laevis/fisiologia , Animais , Western Blotting , Movimento Celular , Ativação do Canal Iônico , Oócitos/citologia , Técnicas de Patch-Clamp/métodos
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