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Fast Inactivation in voltage-gated Na+channels plays essential roles in numerous physiological functions. The canonical hinged-lid model has long predicted that a hydrophobic motif in the DIII-DIV linker (IFM) acts as the gating particle that occludes the permeation pathway during fast inactivation. However, the fact that the IFM motif is located far from the pore in recent high-resolution structures of Nav+channels contradicts thisstatus quomodel. The precise molecular determinants of fast inactivation gate once again, become an open question. Here, we provide a mechanistic reinterpretation of fast inactivation based on ionic and gating current data. In Nav1.4 the actualinactivation gateis comprised of two hydrophobic rings at the bottom of S6. These functionin seriesand closing once the IFM motif binds. Reducing the volume of the sidechain in both rings led to a partially conductive inactivated state. Our experiments also point to a previously overlooked coupling pathway between the bottom of S6 and the selectivity filter.

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 10 ºC in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with submillisecond time resolution by measuring the time-course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir 1.1b, 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 proteins thermodynamics.

Significance Modulation of cellular electrophysiology helps develop an understanding of cellular development and function in healthy and diseased states. We modulate the electrophysiology of neuronal cells in two-dimensional (2D) and 3D assemblies with subcellular precision via photothermal stimulation using a multiscale fuzzy graphene nanostructure. Nanowire (NW)-templated 3D fuzzy graphene (NT-3DFG) nanostructures enable remote, nongenetic photothermal stimulation with laser energies as low as subhundred nanojoules without generating cellular stress. NT-3DFG serves as a powerful toolset for studies of cell signaling within and between in vitro 3D models (human-based organoids and spheroids) and can enable therapeutic interventions., 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.

The observation that membrane capacitance increases with temperature has led to the development of new methods of neuronal stimulation using light. The optocapacitive effect refers to a light-induced change in capacitance produced by the heating of the membrane through a photothermal effect. This change in capacitance manifests as a current, named optocapacitive current that depolarizes cells and therefore can be used to stimulate excitable tissues. Here, we discuss how optocapacitance arises from basic membrane properties, the characteristics of the optocapacitive current, its use for neuronal stimulation, and the challenges for its application in vivo.

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.

The ability to modulate cellular electrophysiology is a fundamental aspect for investigation of tissue development, maturation and function. Currently, there is a need for remote, non-genetic, light-induced control of cellular activity in native-like state, three-dimensional (3D) tissues such as spheroids and organoids. Here, we report a breakthrough hybrid-nanomaterial for remote, non-genetic photostimulation of both two-dimensional (2D) and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photostimulation at subcellular precision with laser energies lower than hundred nanojoules, 1-2 orders of magnitude lower than Au-, C- and Si-based photostimulation. Photostimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves a novel powerful toolset for studies of cell signaling within and between tissues and can enable new therapeutic interventions.

The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca2+) and membrane voltage. Electrophysiological and atomic force microscopy experiments indicate that Ca2+ stabilizes the hemichannel closed state. However, structural data show that Ca2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca2+ inhibits the water flux through Cx hemichannels. We conclude that Ca2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.

Several mutations in Cx26 promote keratitis ichthyosis deafness (KID), a rare disease in which patients exhibit deafness, skin and corneal disorders. Some KID mutations are located in the amino terminal segment of Cx26, which has been proposed as the putative voltage sensor. Most syndromic mutations elicit a gain of hemichannel function, however the mechanism underlying this phenomenon remains unclear. Here we examined the biophysical properties of the mutant G12R at macroscopic and single channel levels. We perform two-electrode voltage clamp to study the deactivation kinetics and apparent affinity for calcium of hemichannel currents. In contrast to wild-type (wt) Cx26, we found that the activation kinetics of the mutant lack saturation of the current upon depolarization pulses above +40 mV. Estimates of the deactivation time constant yield values of 5s and 10s for G12R and wtCx26 hemichannels, respectively; however the apparent affinity for calcium remains similar to wtCx26. Single channel recordings analysis showed that the mutant virtually eliminates the transitions to the subconductance state. Moreover, although the single channel conductance was similar to wtCx26 hemichannels (320 pS), the open probability and mean open times of G12R hemichannels become larger. Molecular dynamics indicates that the G12R induce an electrostatic effect and ddG calculations support protein destabilization.Supported by Fondecyt-3150634 to IEG; Fondecyt-3140590 to GC; Fondecyt-1130855 to ADM; Anillo-ACT1104 to ADM and CG; Fondecyt-1120802 to CG; Grant RO1-GM099490 to J.E.C; AP has a Conicyt Fellowship. The Centro Interdisciplinario de Neurociencias de Valparaiso is a Chilean Millennium Institute (P09-022-F).

Indexación: Scopus. Connexin-based channels comprise hemichannels and gap junction channels. The opening of hemichannels allow for the flux of ions and molecules from the extracellular space into the cell and vice versa. Similarly, the opening of gap junction channels permits the diffusional exchange of ions and molecules between the cytoplasm and contacting cells. The controlled opening of hemichannels has been associated with several physiological cellular processes; thereby unregulated hemichannel activity may induce loss of cellular homeostasis and cell death. Hemichannel activity can be regulated through several mechanisms, such as phosphorylation, divalent cations and changes in membrane potential. Additionally, it was recently postulated that redox molecules could modify hemichannels properties in vitro. However, the molecular mechanism by which redox molecules interact with hemichannels is poorly understood. In this work, we discuss the current knowledge on connexin redox regulation and we propose the hypothesis that extracellular cysteines could be important for sensing changes in redox potential. Future studies on this topic will offer new insight into hemichannel function, thereby expanding the understanding of the contribution of hemichannels to disease progression. http://journal.frontiersin.org/article/10.3389/fphys.2016.00001/full

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.

Salmonella enterica serotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported that S. Gallinarum harbors a type VI secretion system (T6SS) encoded in Salmonella pathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observed in vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced under in vitro bacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins. In vitro bacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon after Salmonella uptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpV and vgrG) revealed that SPI-19 T6SS contributes to S. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked to Salmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used by Salmonella to survive within host cells.

Hemichannels (HCs) and gap junction channels (GJCs) formed by protein subunits called connexins (Cxs) are major pathways for intercellular communication. While HCs connect the intracellular compartment with the extracellular milieu, GJCs allow the interchange of molecules between cytoplasm of two contacting cells. Under physiological conditions, HCs are mostly closed, but they can open under certain stimuli allowing the release of autocrine and paracrine molecules. Moreover, some pathological conditions, like ischemia or other inflammation conditions, significantly increase HCs activity. In addition, some mutations in Cx genes associated with human diseases, such as deafness or cataracts, lead to the formation of more active HCs or “leaky HCs”. In this article we will revise cellular and molecular mechanisms underlying the appearance of leaky HCs, and the consequences of their expression in different cellular systems and animal models, in seeking a common pattern or pathological mechanism of disease.

The most well-known role of Connexins (Cxs) is to communicate the cytoplasm of two adjacent cells forming a gap junction channel in the apposition zone. On the other hand, Cxs form the so called “hemichannels”, a fully functional voltage-gated non-selective ion channel, which present two gating mechanism called “slow” and “fast” gating. Hemichannels consist of 6 monomers, each one composed of four transmembrane domains. Despite the fact that hemichannels are voltage-activated, they lack the classical voltage-sensing domain described for canonical voltage-gated channels. Furthermore, the molecular determinants associated to the voltage activation in connexins hemichannels are still elusive. In this work we search for the molecular determinants associated to voltage detection in human Cx26 hemichannels. Using two electrode voltage-clamp in Xenopus laevis oocytes, we study how the steady-state conductance-voltage (G/V) relationship and kinetics of gating are affected by the neutralization of different charged residues located within the first transmembrane domain. We have found that Cx26 conductance-voltage curve shows an apparent number of gating charges (zδ) of 1.4 e0 for the slow gating mechanism. On the other hand, we obtained a zδ of 3.9 e0 for the fast gate, which is in agreement with the zδ of Cx26 gap junctions. A simple three-state closed-open1-open2 kinetic model is able to account for the channel voltage dependence. Furthermore, our data shows that neutralizing the K41 residue (K41N) increases the zδ; of the slow gate to 2.5 e0, and decreases the zδ of the fast gate to 2.3 e0.This work is supported by FONDECYT Grants 1110430 (R.L.), 1120802 (C.G.), 1130855 (A.M.); ANILLO Grant ACT1104 (C.G.); Beca para Estudios de Magister en Chile Ano 2014 (B.P.); CINV is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economia, Fomento y Turismo.

Connexins (Cxs) are transmembrane proteins involved in the electrical coupling of cells, the release of signaling molecules, and cell proliferation among others. These proteins are expressed in almost every tissue of the human body and mutations in these proteins are related to several hereditary diseases. Cxs monomers oligomerize in hexamers, which traffic to the plasma membrane and form connexons or hemichannels that can act as plasma membrane channels or can travel to intercellular contact zones where they form intercellular channels known as gap junctions. The opening of the Cx hemichannels in the membrane is tightly regulated by Ca2+ and membrane voltage. This regulation prevents leakage of cellular content and its malfunction may lead to pathologic conditions. In this work we studied the reciprocal regulation of Cxs hemichannels by Ca2+ and voltage. We expressed Cx46 in Xenopus laevis oocytes, and using two electrode voltage clamp, analyzed the inhibition of currents by Ca2+. We observed that Cxs inhibition by Ca2+ is voltage dependent. The apparent Ca2+ sensitivity is increased as the membrane voltage is made more negative. This change in the affinity can be explained by a stabilization of the closed state of the Cx channel by Ca2+. We propose a model in which the calcium-bonded state prevents the opening of the hemichannel. The model allows us to determine the affinity of the closed state for calcium. In agreement with the model and suggesting a stabilization of a conformational change that narrows the pore, calcium inhibits the water flux trough Cx hemichannels. This work supports the idea that Ca2+ and voltage act synergistically to promote the closing of the pore in Cx channels.

Salmonella enterica serotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported that S . Gallinarum harbors a type VI secretion system (T6SS) encoded in Salmonella pathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observed in vivo . Expression analyses revealed that SPI-19 T6SS core components are expressed and produced under in vitro bacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins. In vitro bacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon after Salmonella uptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components ( clpV and vgrG ) revealed that SPI-19 T6SS contributes to S . Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked to Salmonella -induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used by Salmonella to survive within host cells.