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Results for “Membrane Potentials”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A red-emitting carborhodamine for monitoring and measuring membrane potential

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Development of Lipid-Coated Semiconductor Nanosensors for Recording of Membrane Potential in Neurons

In the past decade, optical imaging methods have significantly improved our understanding of the information processing principles in the brain. Although many promising tools have been designed, sensors of membrane potential are lagging behind the rest. Semiconductor nanoparticles are an attractive alternative to classical voltage indicators, such as voltage-sensitive dyes and proteins. Such nanoparticles exhibit high sensitivity to external electric fields via the quantum-confine Stark effect. Here we report the development of semiconductor voltage-sensitive nanorods (vsNRs) that self-insert into the neuronal membrane. To facilitate interaction of the nanorods with the membrane, we functionalized their surface with the lipid mixture derived from brain extract. We describe a workflow to detect and process the photoluminescent signal of vsNRs after widefield time-lapse recordings. We also present data indicating that vsNRs are feasible for sensing membrane potential in neurons at a single-particle level. This shows the potential of vsNRs for the detection of neuronal activity with unprecedentedly high spatial and temporal resolution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Wireless Bioelectronic Modulation of Membrane Potential in Glioblastoma Using Carbon Nanotube Porins

Disruption of membrane potential (V mem ) can activate pathways associated with cancer proliferation. Manipulating ion channels may therefore present an effective strategy for treating cancers that fail to respond to conventional therapies. One approach to target these channels is to manipulate the membrane charge, which involves the use of wireless bipolar electrodes such as carbon nanotube porins (CNTPs) inserted into cell membranes to modulate membrane charge and ionic flux. By utilizing membrane dyes, we observed alterations in V mem induced by CNTPs and externally applied voltages. Analyses of cellular behaviors and processes indicated that V mem is more receptive to stimuli in invasive cancers, while it leads to increased metabolism in less invasive cancers, with notable changes in the cell cycle occurring at approximately 48 h post-treatment in Glioblastoma (GB) cell lines. This work shows that CNTPs, in combination and with externally applied voltages, can modulate V mem and alter cancer cell processes, supporting their potential as a therapeutic.

bioelectricity↗
DOE OSTI2020

Kv2.1 channels play opposing roles in regulating membrane potential, Ca 2+ channel function, and myogenic tone in arterial smooth muscle

The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K + channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type Ca V 1.2 channels, lowering intracellular Ca 2+ ([Ca 2+ ] i ) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of Ca V 1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of Ca V 1.2–Ca V 1.2 interactions within these clusters, Kv2.1 increases Ca 2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger Ca V 1.2 clusters, larger [Ca 2+ ] i , and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not Ca V 1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and Ca V 1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca 2+ ] i , and myogenic tone in arterial myocytes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Point-localized, site-specific membrane potential optical recording by single fluorescent nanodiscs

Nanodisc technology was implemented as a platform for voltage nanosensors. A fluorescence (Förster) resonance energy transfer (FRET)-based voltage-sensing scheme employing fluorescent nanodiscs and the hydrophobic ion dipicrylamine was developed and utilized to optically record membrane potentials on the single-nanodisc level. Ensemble and single-nanosensor recordings were demonstrated for HEK293 cells and primary cortical neuron cells. Conjugation of nanodiscs to anti-GABA A antibodies allowed for site-specific membrane potential measurements from postsynaptic sites.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2022

Membrane potential sensing: Material design and method development for single particle optical electrophysiology

We review the development of “single” nanoparticle-based inorganic and organic voltage sensors, which can eventually become a viable tool for “non-genetic optogenetics.” The voltage sensing is accomplished with optical imaging at the fast temporal response and high spatial resolutions in a large field of view. Inorganic voltage nanosensors utilize the Quantum Confined Stark Effect (QCSE) to sense local electric fields. Engineered nanoparticles achieve substantial single-particle voltage sensitivity (~2% Δλ spectral Stark shift up to ~30% ΔF/F per 160 mV) at room temperature due to enhanced charge separation. A dedicated home-built fluorescence microscope records spectrally resolved images to measure the QCSE induced spectral shift at the single-particle level. Biomaterial based surface ligands are designed and developed based on theoretical simulations. The hybrid nanobiomaterials satisfy anisotropic facet-selective coating, enabling effective compartmentalization beyond non-specific staining. Self-spiking- and patched-HEK293 cells and cortical neurons, when stained with hybrid nanobiomaterials, show clear photoluminescence intensity changes in response to membrane potential (MP) changes. Organic voltage nanosensors based on polystyrene beads and nanodisk technology utilize Fluorescence (Förster) Resonance Energy Transfer (FRET) to sense local electric fields. Voltage sensing FRET pairs achieve voltage sensitivity up to ~35% ΔF/F per 120 mV in cultures. Non-invasive MP recording from individual targeted sites (synapses and spines) with nanodisks has been realized. However, both of these QCSE- and FRET-based voltage nanosensors yet need to reach the milestone of recording individual action potentials from individual targeted sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

TrkA undergoes a tetramer-to-dimer conversion to open TrkH which enables changes in membrane potential

TrkH is a bacterial ion channel implicated in K + uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.

36 MATERIALS SCIENCE↗
NASA NTRS2002

The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

Shariff, Karim↗
NASA NTRS2004

The force exerted by the membrane potential during protein import into the mitochondrial matrix

The force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated on the basis of continuum electrostatics. The force is found to vary from 3.0 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 6.5 to 12 A, its measured range. In the present model, the decrease in force with increasing pore width arises from the shielding effect of water. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a purely membranous pore without water gives a force of 3.2 pN per unit charge, which should represent an upper limit. When applied to mitochondrial import experiments on the protein barnase, these results imply that forces between 11 +/- 2 pN and 13.5 +/- 2.5 pN catalyze the unfolding of barnase in those experiments. A comparison of these results with unfolding forces measured using atomic force microscopy is made.

Models, Molecular↗
NASA NTRS1983

ECUT: Energy Conversion and Utilization Technologies program biocatalysis research activity. Potential membrane applications to biocatalyzed processes: Assessment of concentration polarization and membrane fouling

Separation and purification of the products of biocatalyzed fermentation processes, such as ethanol or butanol, consumes most of the process energy required. Since membrane systems require substantially less energy for separation than most alternatives (e.g., distillation) they have been suggested for separation or concentration of fermentation products. This report is a review of the effects of concentration polarization and membrane fouling for the principal membrane processes: microfiltration, ultrafiltration, reverse osmosis, and electrodialysis including a discussion of potential problems relevant to separation of fermentation products. It was concluded that advanced membrane systems may result in significantly decreased energy consumption. However, because of the need to separate large amounts of water from much smaller amounts of product that may be more volatile than wate, it is not clear that membrane separations will necessarily be more efficient than alternative processes.

Ingham, J. D.↗
DOE OSTI2022

Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Review, Tutorial↗
DOE OSTI2022

Bacterial Homologs of Progestin and AdipoQ Receptors (PAQRs) Affect Membrane Energetics Homeostasis but Not Fluidity

Membrane potential homeostasis is essential for cell survival. Defects in membrane potential lead to pleiotropic phenotypes, consistent with the central role of membrane energetics in cell physiology. Homologs of the progestin and AdipoQ receptors (PAQRs) are conserved in multiple phyla of Bacteria and Eukarya. In eukaryotes, PAQRs are proposed to modulate membrane fluidity and fatty acid (FA) metabolism. The role of bacterial homologs has not been elucidated. Here, we use Escherichia coli and Bacillus subtilis to show that bacterial PAQR homologs, which we name “TrhA,” have a role in membrane energetics homeostasis. Using transcriptional fusions, we show that E. coli TrhA (encoded by yqfA) is part of the unsaturated fatty acid biosynthesis regulon. Fatty acid analyses and physiological assays show that a lack of TrhA in both E. coli and B. subtilis (encoded by yplQ) provokes subtle but consistent changes in membrane fatty acid profiles that do not translate to control of membrane fluidity. Instead, membrane proteomics in E. coli suggested a disrupted energy metabolism and dysregulated membrane energetics in the mutant, though it grew similarly to its parent. These changes translated into a disturbed membrane potential in the mutant relative to its parent under various growth conditions. Similar dysregulation of membrane energetics was observed in a different E. coli strain and in the distantly related B. subtilis. Together, our findings are consistent with a role for TrhA in membrane energetics homeostasis, through a mechanism that remains to be elucidated.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Stress-regulated Arabidopsis GAT2 is a low affinity γ-aminobutyric acid transporter

Abstract The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K0.5GABA ~8 mM. l-Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2-overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress.

Plant Sciences↗