RESUMEN
The conventional microelectrodes for recording neuronal activities do not have innate selectivity to cell type, which is one of the critical limitations for the detailed analysis of neuronal circuits. In this study, we engineered a downsized variant of the artificial synapse organizer based on neurexin1ß and a peptide-tag, fabricated gold microelectrodes functionalized with the receptor for the organizer, and performed validation experiments in primary cultured neurons. Successful inductions of synapse-like junctions were detected at the sites of contact between neurons expressing the engineered synapse organizer and functionalized microelectrodes, but not in the negative control experiment in which the electrode functionalization was omitted. Such a molecularly inducible neuron-microelectrode junction could be the basis for the next-generation electrophysiological technique enabling cell type-selective recording.
Asunto(s)
Microelectrodos , Neuronas , Sinapsis , Animales , Neuronas/metabolismo , Sinapsis/metabolismo , Sinapsis/fisiología , Células Cultivadas , Ratas , Ingeniería de Proteínas/métodosRESUMEN
BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. RESULTS: We detected robust voltage-dependent signals from widely distributed positions including N-terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage-sensor domain in the down-state. We also found that the mechanical coupling between the voltage-sensor and phosphatase domains affects the depolarization-induced optical signals but not the hyperpolarization-induced signals. CONCLUSIONS: These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage-sensor domain transition as well as its coupling with the effector.
Asunto(s)
Potenciales de la Membrana , Animales , Potenciales de la Membrana/fisiología , Oocitos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Citoplasma/metabolismo , Xenopus laevis , Dominios Proteicos , Técnicas de Placa-ClampRESUMEN
It has been proposed that cell-type-specific bioelectronic interfaces for neuronal circuits could be established by utilizing the function of synapse organizers. For this purpose, using neurexin-1ß and a peptide tag, we engineered compact synapse organizers that do not interact with the naturally occurring receptors but induce presynaptic differentiation upon contact with nanobody-decorated objects in cultured mammalian and chick forebrain neurons. In chick neurons, the engineered organizer exerted synaptogenesis typically in â¼4 h after the contact, even under an air atmosphere at room temperature, thereby providing a useful cellular model for establishing the molecularly inducible neuron-microelectrode interface.
Asunto(s)
Neuronas , Sinapsis , Animales , Microelectrodos , Sinapsis/fisiología , Diferenciación Celular , Péptidos , MamíferosRESUMEN
Reverse pH-dependent fluorescent protein, including dKeima, is a type of fluorescent protein in which the chromophore protonation state depends inversely on external pH. The dependence is maintained even when immobilized at the metal-solution interface. But, interestingly, its responses to the hydrogen evolution reaction (HER) at the interface are not reversed: HER rises the pH of the solution around the cathode, but, highly active HER induces chromophore deprotonation regardless of the reverse pH dependence, reflecting an interface-specific deprotonation effect by HER. Here, we exploit this phenomenon to perform scanning-less, real-time visualization of interfacial proton dynamics during HER at a wide field of view. By using dKeima, the HER-driven deprotonation effect was well discriminated from the solution pH effect. In the electrodes of composite structures with a catalyst, dKeima visualized keen dependence of the proton depletion pattern on the electrode configuration. In addition, propagations of optical signals were observed, which seemingly reflect long-range proton hopping confined to the metal-solution interface. Thus, reverse pH-dependent fluorescent proteins provide a unique tool for spatiotemporal analysis of interfacial proton dynamics, which is expected to contribute to a better understanding of the HER process and ultimately to the safe and efficient production of molecular hydrogen.
Asunto(s)
Hidrógeno , Protones , Hidrógeno/química , Fluorescencia , Concentración de Iones de Hidrógeno , Proteínas Fluorescentes Verdes/químicaRESUMEN
Clustering of neurexin-1ß occurs through the formation of a trans-cellular complex with neuroligin-1, which promotes the generation of presynapse. While the extracellular region of neurexin-1ß functions to constitute the heterophilic binding interface with neuroligin-1, it has remained unclear whether the region could also play any key role in exerting the intracellular signaling for presynaptic differentiation. In this study, we generated neurexin-1ß lacking the binding site to neuroligin-1 and with a FLAG epitope at the N-terminus, and examined its activity in cultured neurons. The engineered protein still exhibited robust synaptogenic activities upon the epitope-mediated clustering, indicating that the region for complex formation and that for transmitting presynapse differentiation signals are structurally independent of each other. Using a fluorescence protein as an epitope, synaptogenesis was also induced by a gene-codable nanobody. The finding opens possibilities of neurexin-1ß as a platform for developing various molecular tools which may allow, for example, precise modifications of neural wirings under genetic control.
Asunto(s)
Moléculas de Adhesión Celular Neuronal , Sinapsis , Epítopos/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Sinapsis/metabolismo , Neuronas/metabolismo , Unión ProteicaRESUMEN
Voltage sensing phosphatase (VSP), consists of a voltage sensor domain (VSD) like that found in voltage-gated ion channels and a phosphoinositide (PIP) phosphatase region exhibiting remarkable structural similarity to a tumor suppressor enzyme, PTEN. Membrane depolarization activates the enzyme activity through tight coupling between the VSD and enzyme region. The VSD of VSP has a unique nature; it is a self-contained module that can be transferred to other proteins, conferring voltage sensitivity. Thanks to this nature, numerous versions of gene-encoded voltage indicators (GEVIs) have been developed through combination of a fluorescent protein with the VSD of VSP. In addition, VSP itself can also serve as a tool to alter PIP levels in cells. Cellular levels of PIPs, PI(4,5)P2 in particular, can be acutely and transiently reduced using a simple voltage protocol after heterologous expression of VSP. Recent progress in our understanding of the molecular structure and mechanisms underlying VSP facilitates optimization of its molecular properties for its use as a molecular tool.
Asunto(s)
Fosfatidilinositoles , Monoéster Fosfórico Hidrolasas , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Predation pressure occurs as a result of predation frequency and prey vulnerability. Although quantifying these factors individually is essential to precisely understand predation effects on evolution, they have been generally less accessible. Here, using a modified form of Poisson function, we quantified the frequencies and vulnerabilities, as well as the resulting predation pressures, concerning the shell drillers versus prey interactions from the Eocene and Miocene periods. Our analysis quantitatively revealed that low-spired shells tend to show increased vulnerability except for two planispiral species that exhibit an unexpectedly low vulnerability. We then identified septal structures within the two species that resemble those in nautiloids and ammonoids but which provided a defensive role against the predators, enhancing the mean lifetime by approximately 20%. The current approach enables us to quantitatively trace how predation frequency and prey vulnerability have interacted, been transformed spatio-temporally, and been a driving force of evolution at geological time scales.
RESUMEN
Cation channel of Spermatozoa (CatSper) is one of the voltage-gated ion channels consisting of voltage sensor domains (VSDs) and pore-gate domains. CatSper is exclusively expressed in spermatozoa and indispensable for Ca2+ influx into cytosol. Recently, we have reported that the VSD of ascidian CatSper induces Ca2+-permeable pathways in heterologous expression systems. However, it is not known whether ion permeability through the VSD of CatSper is conserved in mammals. In the present study, electrophysiology and fluorometry in Xenopus oocytes revealed that Ca2+-permeable paths are also formed by expressing the VSD of murine CatSper. We also examined the permeability to monovalent cations other than Na+ in the VSD of ascidian CatSper.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Espermatozoides/metabolismo , Animales , Masculino , Ratones , Xenopus laevisRESUMEN
Ion channel activity in the plasma membrane of living cells generates voltage changes that are critical for numerous biological functions. The membrane of the endoplasmic/sarcoplasmic reticulum (ER/SR) is also endowed with ion channels, but whether changes in its voltage occur during cellular activity has remained ambiguous. This issue is critical for cell functions that depend on a Ca2+ flux across the reticulum membrane. This is the case for contraction of striated muscle, which is triggered by opening of ryanodine receptor Ca2+ release channels in the SR membrane in response to depolarization of the transverse invaginations of the plasma membrane (the t-tubules). Here, we use targeted expression of voltage-sensitive fluorescence resonance energy transfer (FRET) probes of the Mermaid family in differentiated muscle fibers to determine whether changes in SR membrane voltage occur during depolarization-contraction coupling. In the absence of an SR targeting sequence, FRET signals from probes present in the t-tubule membrane allow calibration of the voltage sensitivity and amplitude of the response to voltage-clamp pulses. Successful SR targeting of the probes was achieved using an N-terminal domain of triadin, which completely eliminates voltage-clamp-activated FRET signals from the t-tubule membrane of transfected fibers. In fibers expressing SR-targeted Mermaid probes, activation of SR Ca2+ release in the presence of intracellular ethyleneglycol-bis(ß-amino-ethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) results in an accompanying FRET signal. We find that this signal results from pH sensitivity of the probe, which detects cytosolic acidification because of the release of protons upon Ca2+ binding to EGTA. When EGTA is substituted with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the contraction blocker N-benzyl-p-toluene sulfonamide, we find no indication of a substantial change in the FRET response caused by a voltage change. These results suggest that the ryanodine receptor-mediated SR Ca2+ efflux is well balanced by concomitant counterion currents across the SR membrane.
Asunto(s)
Fibras Musculares Esqueléticas/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Técnicas In Vitro , Masculino , Ratones , Antígenos de Histocompatibilidad Menor , Proteínas de Complejo Poro Nuclear , Técnicas de Placa-ClampRESUMEN
Multisensory integration (MSI) is a fundamental emergent property of the mammalian brain. During MSI, perceptual information encoded in patterned activity is processed in multimodal association cortex. The systems-level neuronal dynamics that coordinate MSI, however, are unknown. Here, we demonstrate intrinsic hub-like network activity in the association cortex that regulates MSI. We engineered calcium reporter mouse lines based on the fluorescence resonance energy transfer sensor yellow cameleon (YC2.60) expressed in excitatory or inhibitory neurons. In medial and parietal association cortex, we observed spontaneous slow waves that self-organized into hubs defined by long-range excitatory and local inhibitory circuits. Unlike directional source/sink-like flows in sensory areas, medial/parietal excitatory and inhibitory hubs had net-zero balanced inputs. Remarkably, multisensory stimulation triggered rapid phase-locking mainly of excitatory hub activity persisting for seconds after the stimulus offset. Therefore, association cortex tends to form balanced excitatory networks that configure slow-wave phase-locking for MSI. VIDEO ABSTRACT.
Asunto(s)
Corteza Cerebral/fisiología , Neuronas/fisiología , Animales , Corteza Cerebral/citología , RatonesRESUMEN
The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.
Asunto(s)
Canales de Calcio , Cationes Bivalentes/metabolismo , Activación del Canal Iónico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Animales , Canales de Calcio/química , Canales de Calcio/genética , Canales de Calcio/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Conductividad Eléctrica , Femenino , Células HEK293 , Humanos , Activación del Canal Iónico/genética , Potenciales de la Membrana/genética , Permeabilidad , Dominios Proteicos/genética , XenopusRESUMEN
We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible.
Asunto(s)
Expresión Génica , Genes Reporteros , Mediciones Luminiscentes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Imagen Molecular , Optogenética , Animales , Línea Celular , Células Cultivadas , Fenómenos Electrofisiológicos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/química , Modelos Moleculares , Imagen Molecular/métodos , Optogenética/métodos , Conformación ProteicaRESUMEN
Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.
Asunto(s)
Técnicas Biosensibles , Potenciales de la Membrana/genética , Miocitos Cardíacos/metabolismo , Potenciales de Acción/genética , Animales , Animales Modificados Genéticamente , Sistema Cardiovascular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Genes Reporteros , Humanos , Proteínas Recombinantes de Fusión/genética , Investigación , Imagen de Colorante Sensible al VoltajeRESUMEN
Crystallization of proteins may occur in the cytosol of a living cell, but how a cell responds to intracellular protein crystallization remains unknown. We developed a variant of coral fluorescent protein that forms diffraction-quality crystals within mammalian cells. This expression system allowed the direct determination of its crystal structure at 2.9 Å, as well as observation of the crystallization process and cellular responses. The micron-sized crystal, which emerged rapidly, was a pure assembly of properly folded ß-barrels and was recognized as an autophagic cargo that was transferred to lysosomes via a process involving p62 and LC3. Several lines of evidence indicated that autophagy was not required for crystal nucleation or growth. These findings demonstrate that in vivo protein crystals can provide an experimental model to study chemical catalysis. This knowledge may be beneficial for structural biology studies on normal and disease-related protein aggregation.
Asunto(s)
Antozoos/química , Citosol/metabolismo , Proteínas Fluorescentes Verdes/química , Lisosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Autofagia , Cristalización , Cristalografía por Rayos X , Citosol/ultraestructura , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Lisosomas/ultraestructura , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Neuronas/metabolismo , Neuronas/ultraestructura , Cultivo Primario de Células , Pliegue de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Sequestosoma-1 , Difracción de Rayos XRESUMEN
Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its ß-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential.
RESUMEN
Voltage-sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage-gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage-dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn-VSP) and Cynops VSP (Cp-VSP), including the positively-charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C-terminal C2 domain of Hn-VSP is significantly shorter than that of Cp-VSP and other VSP orthologs. RT-PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage-dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed "sensing" currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp-VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn-VSP lacked such phosphatase activity due to the truncation of its C2 domain.
RESUMEN
Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103(+)DCs, including Langerin(+)CD103(+)dermal DCs (DDCs), remained in the dLN for 4-4.5 days after migration from the skin, while CD103(-)DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103(-)DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.
Asunto(s)
Células Dendríticas/citología , Ganglios Linfáticos/citología , Piel/citología , Animales , Antígenos CD/metabolismo , Movimiento Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Dermatitis Irritante/inmunología , Dermatitis Irritante/patología , Técnicas de Sustitución del Gen , Cadenas alfa de Integrinas/metabolismo , Células de Langerhans/citología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Luz , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Proteínas/genética , Receptores CCR7/deficiencia , Receptores CCR7/genética , Receptores CCR7/metabolismo , Piel/inmunología , Piel/metabolismoRESUMEN
The development of a high performance protein probe for the measurement of membrane potential will allow elucidation of spatiotemporal regulation of electrical signals within a network of excitable cells. Engineering such a probe requires a functional screen of many candidates. Although the glass-microelectrode technique generally provides an accurate measure of a given test probe, throughputs are limited. In this study, we focused on an approach that uses the membrane potential changes induced by an external electric field in a geometrically simple mammalian cell. For quantitative evaluation of membrane voltage probes that rely on the structural transition of the S1-S4 voltage sensor domain and hence have non-linear voltage dependencies, it was crucial to introduce exogenous inwardly rectifying potassium conductance to reduce cell-to-cell variability in resting membrane potentials. Importantly, the addition of the exogenous conductance drastically altered the profile of the field-induced potential. Following a site-directed random mutagenesis and the rapid screen, we identified a mutant of a voltage probe Mermaid, exhibiting positively shifted voltage sensitivity. Due to its simplicity, the current approach will be applicable under a microfluidic configuration to carry out an efficient screen. Additionally, we demonstrate another interesting aspect of the field-induced optical signals, ability to visualize electrical couplings between cells.
Asunto(s)
Potenciales de la Membrana/fisiología , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Conductividad Eléctrica , Células HEK293 , Humanos , Ratones , Microelectrodos , Datos de Secuencia Molecular , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Alineación de SecuenciaRESUMEN
One of the most awaited techniques in modern physiology is the sensitive detection of spatiotemporal electrical activity in a complex network of excitable cells. The use of genetically encoded voltage probes has been expected to enable such analysis. However, in spite of recent progress, existing probes still suffer from low signal amplitude and/or kinetics too slow to detect fast electrical activity. Here, we have developed an improved voltage probe named Mermaid2, which is based on the voltage-sensor domain of the voltage-sensing phosphatase from Ciona intestinalis and Förster energy transfer between a pair of fluorescent proteins. In mammalian cells, Mermaid2 permits ratiometric readouts of fractional changes of more than 50% over a physiologically relevant voltage range with fast kinetics, and it was used to follow a train of action potentials at frequencies of up to 150 Hz. Mermaid2 was also able to detect single action potentials and subthreshold voltage responses in hippocampal neurons in vitro, in addition to cortical electrical activity evoked by sound stimuli in single trials in living mice.
Asunto(s)
Potenciales de Acción , Neuroimagen Funcional/métodos , Optogenética/métodos , Monoéster Fosfórico Hidrolasas/genética , Animales , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Neuronas/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , XenopusRESUMEN
The voltage-sensor domain (VSD) is a functional module that undergoes structural transitions in response to membrane potential changes and regulates its effectors, thereby playing a crucial role in amplifying and decoding membrane electrical signals. Ion-conductive pore and phosphoinositide phosphatase are the downstream effectors of voltage-gated channels and the voltage-sensing phosphatase, respectively. It is known that upon transition, the VSD generally acts on the region C-terminal to S4. However, whether the VSD also induces any structural changes in the N-terminal region of S1 has not been addressed directly. Here, we report the existence of such an N-terminal effect. We used two distinct optical reporters-one based on the Förster resonance energy transfer between a pair of fluorescent proteins, and the other based on fluorophore-labeled HaloTag-and studied the behavior of these reporters placed at the N-terminal end of the monomeric VSD derived from voltage-sensing phosphatase. We found that both of these reporters were affected by the VSD transition, generating voltage-dependent fluorescence readouts. We also observed that whereas the voltage dependencies of the N- and C-terminal effects appear to be tightly coupled, the local structural rearrangements reflect the way in which the VSD is loaded, demonstrating the flexible nature of the VSD.