RESUMO
Perturbing the temperature of a system modifies its energy landscape, thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage clamp while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 9°C in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with sub-ms time resolution by measuring the time course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir1.1 isoform b, which reveals a highly temperature-sensitive blockage relief, and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane protein thermodynamics.
Assuntos
Canais Iônicos , Oócitos , Animais , Temperatura , Potenciais da Membrana , Canais Iônicos/metabolismo , Membrana Celular/metabolismo , Termodinâmica , Xenopus laevis/metabolismo , Oócitos/metabolismoRESUMO
Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1, encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BKG354S) in KCNMA1, in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BKG354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BKG354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BKG354S channel, but not the BKWT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.
Assuntos
Cerebelo/patologia , Clorzoxazona/administração & dosagem , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Mitocôndrias/patologia , Degenerações Espinocerebelares/genética , Adolescente , Animais , Animais Recém-Nascidos , Linhagem Celular , Cerebelo/citologia , Análise Mutacional de DNA , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mutação com Perda de Função , Camundongos , Oócitos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/tratamento farmacológico , Degenerações Espinocerebelares/patologia , Transfecção , Sequenciamento do Exoma , XenopusRESUMO
The ability to modulate cellular electrophysiology is fundamental to the investigation of development, function, and disease. Currently, there is a need for remote, nongenetic, light-induced control of cellular activity in two-dimensional (2D) and three-dimensional (3D) platforms. Here, we report a breakthrough hybrid nanomaterial for remote, nongenetic, photothermal stimulation of 2D and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photothermal stimulation at subcellular precision without the need for genetic modification, with laser energies lower than a hundred nanojoules, one to two orders of magnitude lower than Au-, C-, and Si-based nanomaterials. Photothermal stimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves as a powerful toolset for studies of cell signaling within and between tissues and can enable therapeutic interventions.
Assuntos
Grafite/química , Nanoestruturas/química , Neurônios/efeitos da radiação , Animais , Técnicas Eletroquímicas , Lasers , Nanofios/química , Neurônios/fisiologia , Processos Fotoquímicos , Ratos , Esferoides Celulares/fisiologia , Esferoides Celulares/efeitos da radiaçãoRESUMO
The activation of voltage-dependent ion channels is associated with the movement of gating charges, which give rise to gating currents. Although gating currents from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents from a population of channels allows a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a rate model of channel activation and assuming sufficiently high bandwidth, is in accordance with the presence of a main step along the activation pathway carrying a charge of 2.3-2.4 e0. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we used a Brownian model of voltage-dependent gating based on atomic detail structure, that follows the laws of electrodynamics. The model predicts gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of the model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real channels the voltage sensor moves along a sequence of intermediate states separated by relatively low (<5 kT) energy barriers. As a consequence, crossings of successive gating charges through the gating pore become very frequent, and the corresponding current shots are often seen to overlap because of the relatively high filtering. Notably, this limited bandwidth effect is at the origin of the relatively high single-step charge experimentally detected.
Assuntos
Ativação do Canal Iônico , Canais IônicosRESUMO
Voltage-gated ion channels play important roles in physiological processes, especially in excitable cells, in which they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared; the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, the possible helix transition from α- to 310-helix in S4 during the activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, in i, i + 3 and i, i + 6 or i, i + 4 and i, i + 7 pairs, to favor 310- or α-helical conformations, respectively. We have taken advantage of the ability of His to coordinate Zn2+ to promote metal ion bridges, and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in α-helical conformation). In the presence of 20 µM of Zn2+, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, and the voltage dependence is shifted by 10 mV toward depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in α-helical conformation, a closed (resting or intermediate) state is stabilized rather than destabilizing the open (active) state. Taken together, our data indicates that S4 undergoes α-helical conformation to a short-lived different secondary structure transiently before reaching the active state in the activation process.
Assuntos
Ativação do Canal Iônico , Superfamília Shaker de Canais de Potássio , Histidina , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de ProteínaRESUMO
The Na+/K+-ATPase is a chemical molecular machine responsible for the movement of Na+ and K+ ions across the cell membrane. These ions are moved against their electrochemical gradients, so the protein uses the free energy of ATP hydrolysis to transport them. In fact, the Na+/K+-ATPase is the single largest consumer of energy in most cells. In each pump cycle, the protein sequentially exports 3Na+ out of the cell, then imports 2K+ into the cell at an approximate rate of 200 cycles/s. In each half cycle of the transport process, there is a state in which ions are stably trapped within the permeation pathway of the protein by internal and external gates in their closed states. These gates are required to open alternately; otherwise, passive ion diffusion would be a wasteful end of the cell's energy. Once one of these gates open, ions diffuse from their binding sites to the accessible milieu, which involves moving through part of the electrical field across the membrane. Consequently, ions generate transient electrical currents first discovered more than 30 years ago. They have been studied in a variety of preparations, including native and heterologous expression systems. Here, we review three decades' worth of work using these transient electrical signals to understand the kinetic transitions of the movement of Na+ and K+ ions through the Na+/K+-ATPase and propose the significance that this work might have to the understanding of the dysfunction of human pump orthologs responsible for some newly discovered neurological pathologies.
Assuntos
ATPase Trocadora de Sódio-Potássio , Sódio , Biofísica , Humanos , Íons/metabolismo , Cinética , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The ionic gradients across cell membranes generate a transmembrane voltage that regulates the function of numerous membrane proteins such as ion channels, transporters, pumps and enzymes. The mechanisms by which proteins sense voltage is diverse: ion channels have a conserved, positively charged transmembrane region that moves in response to changes in membrane potential, some G-protein coupled receptors possess a specific voltage-sensing motif and some membrane pumps and transporters use the ions that they transport across membranes to sense membrane voltage. Characterizing the general features of voltage sensors might lead to the discovery of further membrane proteins that are voltage regulated.
Assuntos
Potenciais da Membrana , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ativação do Canal Iônico , Proteínas de Membrana/química , Dados de Sequência Molecular , Canais de Potássio/química , Canais de Potássio/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.
Assuntos
Transferência de Energia/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas Musculares/química , Canais de Sódio/química , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Compostos de Boro/química , Cinética , Elementos da Série dos Lantanídeos/química , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Oócitos/química , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Xenopus/genéticaRESUMO
The action potential of nerve and muscle is produced by voltage-sensitive channels that include a specialized device to sense voltage. The voltage sensor depends on the movement of charges in the changing electric field as suggested by Hodgkin and Huxley. Gating currents of the voltage sensor are now known to depend on the movements of positively charged arginines through the hydrophobic plug of a voltage sensor domain. Transient movements of these permanently charged arginines, caused by the change of transmembrane potential V, further drag the S4 segment and induce opening/closing of the ion conduction pore by moving the S4-S5 linker. This moving permanent charge induces capacitive current flow everywhere. Everything interacts with everything else in the voltage sensor and protein, and so it must also happen in its mathematical model. A Poisson-Nernst-Planck (PNP)-steric model of arginines and a mechanical model for the S4 segment are combined using energy variational methods in which all densities and movements of charge satisfy conservation laws, which are expressed as partial differential equations in space and time. The model computes gating current flowing in the baths produced by arginines moving in the voltage sensor. The model also captures the capacitive pile up of ions in the vestibules that link the bulk solution to the hydrophobic plug. Our model reproduces the signature properties of gating current: 1) equality of ON and OFF charge Q in integrals of gating current, 2) saturating voltage dependence in the Q(charge)-voltage curve, and 3) many (but not all) details of the shape of gating current as a function of voltage. Our results agree qualitatively with experiments and can be improved by adding more details of the structure and its correlated movements. The proposed continuum model is a promising tool to explore the dynamics and mechanism of the voltage sensor.
Assuntos
Ativação do Canal Iônico , Potenciais da Membrana , Canais de Sódio Disparados por Voltagem/química , Animais , Humanos , Modelos Teóricos , Simulação de Dinâmica Molecular , Domínios Proteicos , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Animais , Transferência de Energia , Feminino , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Oócitos , Conformação Proteica , Domínios Proteicos , Xenopus laevisRESUMO
Two families of accessory proteins, ß and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Elementos da Série dos Lantanídeos/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Subunidades Proteicas/química , Compostos de Boro/químicaRESUMO
Gating of the mammalian inward rectifier Kir1.1 at the helix bundle crossing (HBC) by intracellular pH is believed to be mediated by conformational changes in the C-terminal domain (CTD). However, the exact motion of the CTD during Kir gating remains controversial. Crystal structures and single-molecule fluorescence resonance energy transfer of KirBac channels have implied a rigid body rotation and/or a contraction of the CTD as possible triggers for opening of the HBC gate. In our study, we used lanthanide-based resonance energy transfer on single-Cys dimeric constructs of the mammalian renal inward rectifier, Kir1.1b, incorporated into anionic liposomes plus PIP2, to determine unambiguous, state-dependent distances between paired Cys residues on diagonally opposite subunits. Functionality and pH dependence of our proteoliposome channels were verified in separate electrophysiological experiments. The lanthanide-based resonance energy transfer distances measured in closed (pH 6) and open (pH 8) conditions indicated neither expansion nor contraction of the CTD during gating, whereas the HBC gate widened by 8.8 ± 4 Å, from 6.3 ± 2 to 15.1 ± 6 Å, during opening. These results are consistent with a Kir gating model in which rigid body rotation of the large CTD around the permeation axis is correlated with opening of the HBC hydrophobic gate, allowing permeation of a 7 Å hydrated K ion.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Ativação do Canal Iônico , Elementos da Série dos Lantanídeos , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
Millisecond pulses of laser light delivered to gold nanoparticles residing in close proximity to the surface membrane of neurons can induce membrane depolarization and initiate an action potential. An optocapacitance mechanism proposed as the basis of this effect posits that the membrane-interfaced particle photothermally induces a cell-depolarizing capacitive current, and predicts that delivering a given laser pulse energy within a shorter period should increase the pulse's action-potential-generating effectiveness by increasing the magnitude of this capacitive current. Experiments on dorsal root ganglion cells show that, for each of a group of interfaced gold nanoparticles and microscale carbon particles, reducing pulse duration from milliseconds to microseconds markedly decreases the minimal pulse energy required for AP generation, providing strong support for the optocapacitance mechanism hypothesis.
Assuntos
Potenciais de Ação , Lasers , Fenômenos Ópticos , Carbono/química , Gânglios Espinais/citologia , Ouro/química , Nanopartículas Metálicas/química , Neurônios/citologiaRESUMO
This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world.
Assuntos
Comunicação Celular/fisiologia , Polímeros/química , Semicondutores , Propriedades de SuperfícieRESUMO
The voltage-gated proton channel Hv1 plays a critical role in the fast proton translocation that underlies a wide range of physiological functions, including the phagocytic respiratory burst, sperm motility, apoptosis, and metastatic cancer. Both voltage activation and proton conduction are carried out by a voltage-sensing domain (VSD) with strong similarity to canonical VSDs in voltage-dependent cation channels and enzymes. We set out to determine the structural properties of membrane-reconstituted human proton channel (hHv1) in its resting conformation using electron paramagnetic resonance spectroscopy together with biochemical and computational methods. We evaluated existing structural templates and generated a spectroscopically constrained model of the hHv1 dimer based on the Ci-VSD structure at resting state. Mapped accessibility data revealed deep water penetration through hHv1, suggesting a highly focused electric field, comprising two turns of helix along the fourth transmembrane segment. This region likely contains the H(+) selectivity filter and the conduction pore. Our 3D model offers plausible explanations for existing electrophysiological and biochemical data, offering an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement.
Assuntos
Canais Iônicos/metabolismo , Bicamadas Lipídicas , Prótons , Sequência de Aminoácidos , Dimerização , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Dados de Sequência MolecularRESUMO
Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.
Assuntos
Potenciais de Ação , Fenômenos Eletrofisiológicos , Neurônios/citologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Galinhas/genética , Ativação do Canal Iônico , Cinética , Monoéster Fosfórico Hidrolases/química , Xenopus laevisRESUMO
Silicon-based materials have widespread application as biophysical tools and biomedical devices. Here we introduce a biocompatible and degradable mesostructured form of silicon with multi-scale structural and chemical heterogeneities. The material was synthesized using mesoporous silica as a template through a chemical vapour deposition process. It has an amorphous atomic structure, an ordered nanowire-based framework and random submicrometre voids, and shows an average Young's modulus that is 2-3 orders of magnitude smaller than that of single-crystalline silicon. In addition, we used the heterogeneous silicon mesostructures to design a lipid-bilayer-supported bioelectric interface that is remotely controlled and temporally transient, and that permits non-genetic and subcellular optical modulation of the electrophysiology dynamics in single dorsal root ganglia neurons. Our findings suggest that the biomimetic expansion of silicon into heterogeneous and deformable forms can open up opportunities in extracellular biomaterial or bioelectric systems.
RESUMO
Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.
Assuntos
Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/fisiologia , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos/fisiologia , Animais , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.2/química , Modelos Químicos , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Estrutura Secundária de Proteína/fisiologia , Xenopus laevisRESUMO
Magnesium (Mg(2+)) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg(2+) uptake and is self-regulated by intracellular Mg(2+). Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg(2+)-selective channel, fully excluding monovalent cations, and Ca(2+), whereas in absence of Mg(2+), CorA is essentially nonselective, displaying only mild preference against other divalents (Ca(2+) > Mn(2+) > Co(2+) > Mg(2+) > Ni(2)(+)). Selectivity against monovalent cations takes place via Mg(2+) binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca(2+) channel selectivity despite differences in sequence and overall structure.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Magnésio/metabolismo , Modelos Moleculares , Thermotoga maritima/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Biologia Computacional , Primers do DNA/genética , Vetores Genéticos , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Alinhamento de Sequência , Eletricidade Estática , Thermotoga maritima/química , Thermotoga maritima/metabolismo , Xenopus laevisRESUMO
ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.