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Electrically controlling and optically observing the membrane potential of supported lipid bilayers.Biophysical Journal Jul 2022Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors....
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.
Topics: Cell Membrane; Electricity; Lipid Bilayers; Membrane Potentials; Phospholipids
PubMed: 35619563
DOI: 10.1016/j.bpj.2022.05.037 -
Biochemistry. Biokhimiia Oct 2023An overview of current notions on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme... (Review)
Review
An overview of current notions on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase is presented in this work. It is suggested that the main contribution to Δψ formation is made by the movement of H+ across the membrane along the intra-protein hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.
Topics: Membrane Potentials; Cytochrome b Group; Escherichia coli Proteins; Electron Transport Chain Complex Proteins; Cytochromes; Oxidation-Reduction
PubMed: 38105020
DOI: 10.1134/S0006297923100073 -
The Journal of General Physiology May 2020Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides...
Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestinalis VSP. This modified Dr-VSP (CiDr-VSPmChe L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P2 sensitivity of ion channels in living cells.
Topics: Animals; Cell Line; Cytoplasm; HEK293 Cells; Humans; Mammals; Membrane Potentials; Phosphatidylinositol 4,5-Diphosphate; Phosphoric Monoester Hydrolases; Potassium Channels, Voltage-Gated; TRPC6 Cation Channel
PubMed: 32167537
DOI: 10.1085/jgp.201912491 -
Cell Systems May 2020Cellular membrane potential plays a key role in the formation and retrieval of memories in the metazoan brain, but it remains unclear whether such memory can also be...
Cellular membrane potential plays a key role in the formation and retrieval of memories in the metazoan brain, but it remains unclear whether such memory can also be encoded in simpler organisms like bacteria. Here, we show that single-cell-level memory patterns can be imprinted in bacterial biofilms by light-induced changes in the membrane potential. We demonstrate that transient optical perturbations generate a persistent and robust potassium-channel-mediated change in the membrane potential of bacteria within the biofilm. The light-exposed cells respond in an anti-phase manner, relative to unexposed cells, to both natural and induced oscillations in extracellular ion concentrations. This anti-phase response, which persists for hours following the transient optical stimulus, enables a direct single-cell resolution visualization of spatial memory patterns within the biofilm. The ability to encode robust and persistent membrane-potential-based memory patterns could enable computations within prokaryotic communities and suggests a parallel between neurons and bacteria.
Topics: Bacteria; Biofilms; Membrane Potentials; Memory; Microbiota; Models, Theoretical; Optical Phenomena; Potassium Channels; Voltage-Sensitive Dye Imaging
PubMed: 32343961
DOI: 10.1016/j.cels.2020.04.002 -
International Journal of Molecular... Jan 2023Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the... (Review)
Review
Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.
Topics: Membrane Potentials; Cell Membrane; Fluorescent Dyes; Neurons; Optical Imaging
PubMed: 36768759
DOI: 10.3390/ijms24032435 -
Cell Calcium Jul 2022In the strongly polarized membranes of excitable cells, activation of T-type Ca channels (TTCCs) by weak depolarizing stimuli allows the influx of Ca which further... (Review)
Review
In the strongly polarized membranes of excitable cells, activation of T-type Ca channels (TTCCs) by weak depolarizing stimuli allows the influx of Ca which further amplifies membrane depolarization, thus "recruiting" higher threshold voltage-gated channels to promote action potential firing. Nonetheless, TTCCs perform other functions in the plasma membrane of both excitable and non-excitable cells, in which they regulate a number of biochemical pathways relevant for cell cycle and cell fate. Furthermore, data obtained in the last 20 years have shown the involvement of TTCCs in tumor biology, designating them as promising chemotherapeutic targets. However, their activity in the steadily-depolarized membranes of cancer cells, in which most voltage-gated channels are in the inactivated (nonconducting) state, is counter-intuitive. Here we discuss that in cancer cells weak hyperpolarizing stimuli increase the fraction of open TTCCs which, in association with Ca-dependent K channels, may critically boost membrane hyperpolarization and driving force for Ca entry through different voltage-independent Ca channels. Available evidence also shows that TTCCs participate in positive feedback circuits with signaling effectors, which may warrant a switch-like activation of pro-proliferative and pro-survival pathways in spite of their low availability. Unravelling TTCC modus operandi in the context of non-excitable membranes may facilitate the development of novel anticancer approaches.
Topics: Action Potentials; Calcium; Neoplasms
PubMed: 35691056
DOI: 10.1016/j.ceca.2022.102610 -
Molecules (Basel, Switzerland) Jan 2023In the course of action potential firing, all axons and neurons release K from the intra- cellular compartment into the interstitial space to counteract the depolarizing... (Review)
Review
In the course of action potential firing, all axons and neurons release K from the intra- cellular compartment into the interstitial space to counteract the depolarizing effect of Na influx, which restores the resting membrane potential. This efflux of K from axons results in K accumulation in the interstitial space, causing depolarization of the K reversal potential (E), which can prevent subsequent action potentials. To ensure optimal neuronal function, the K is buffered by astrocytes, an energy-dependent process, which acts as a sink for interstitial K, absorbing it at regions of high concentration and distributing it through the syncytium for release in distant regions. Pathological processes in which energy production is compromised, such as anoxia, ischemia, epilepsy and spreading depression, can lead to excessive interstitial K accumulation, disrupting sensitive trans-membrane ion gradients and attenuating neuronal activity. The changes that occur in interstitial [K] resulting from both physiological and pathological processes can be monitored accurately in real time using K-sensitive microelectrodes, an invaluable tool in electrophysiological studies.
Topics: Microelectrodes; Neurons; Membrane Potentials; Axons; Action Potentials; Potassium
PubMed: 36677581
DOI: 10.3390/molecules28020523 -
American Journal of Physiology. Cell... Jun 2022We endeavored to understand the factors determining the peak force-resting membrane potential () relationships of isolated slow-twitch soleus and fast-twitch extensor...
We endeavored to understand the factors determining the peak force-resting membrane potential () relationships of isolated slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles from mice (25°C), especially in relation to fatigue. Interrelationships between intracellular K activity ([Formula: see text]), extracellular K concentration ([K]), resting , action potentials, and force were studied. The large resting variation was mainly due to the variability of [Formula: see text]. Action potential overshoot-resting relationships determined at 4 and 8-10 mM [K] after short (<5 min) and prolonged (>50 min) depolarization periods revealed a constant overshoot from -90 to -70 mV providing a safety margin. Overshoot decline with depolarization beyond -70 mV was less after short than prolonged depolarization. Inexcitable fibers occurred only with prolonged depolarization. The overshoot decline during action potential trains (2 s) exceeded that during short depolarizations. Concomitant lower extracellular [Na] and raised [K] depressed the overshoot in an additive manner and peak force in a synergistic manner. Raised [K]-induced force loss was exacerbated with transverse wire versus parallel plate stimulation in soleus, implicating action potential propagation failure in the surface membrane. Increasing stimulus pulse parameters restored tetanic force at 9-10 mM [K] in soleus but not EDL, indicative of action potential failure within trains. The peak tetanic force-resting relationships (determined with resting from deeper rather than surface fibers) were dynamic and showed pronounced force depression over -69 to -60 mV in both muscle types, implicating that such depolarization contributes to fatigue. The K-Na interaction shifted this relationship toward less depolarized potentials, suggesting that the combined ionic effect is physiologically important during fatigue.
Topics: Animals; Fatigue; Membrane Potentials; Mice; Muscle Contraction; Muscle Fatigue; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Potassium; Sodium
PubMed: 35385328
DOI: 10.1152/ajpcell.00401.2021 -
International Journal of Molecular... Mar 2023Voltage-gated ion channels are integral membrane proteins that respond to changes in membrane potential with rapid variations in membrane permeability to ions [...].
Voltage-gated ion channels are integral membrane proteins that respond to changes in membrane potential with rapid variations in membrane permeability to ions [...].
Topics: Ion Channels; Membrane Potentials; Cell Membrane Permeability; Ions; Drug Design
PubMed: 37047455
DOI: 10.3390/ijms24076484 -
Bioelectrochemistry (Amsterdam,... Dec 2020Conventional electric stimuli of micro- and millisecond duration excite or activate cells at voltages 10-100 times below the electroporation threshold. This ratio is... (Review)
Review
Conventional electric stimuli of micro- and millisecond duration excite or activate cells at voltages 10-100 times below the electroporation threshold. This ratio is remarkably different for nanosecond electric pulses (nsEP), which caused excitation and activation only at or above the electroporation threshold in diverse cell lines, primary cardiomyocytes, neurons, and chromaffin cells. Depolarization to the excitation threshold often results from (or is assisted by) the loss of the resting membrane potential due to ion leaks across the membrane permeabilized by nsEP. Slow membrane resealing and the build-up of electroporation damages prevent repetitive excitation by nsEP. However, peripheral nerves and muscles are exempt from this rule and withstand multiple cycles of excitation by nsEP without the loss of function or signs of electroporation. We show that the damage-free excitation by nsEP may be enabled by the membrane charging time constant sufficiently large to (1) cap the peak transmembrane voltage during nsEP below the electroporation threshold, and (2) extend the post-nsEP depolarization long enough to activate voltage-gated ion channels. The low excitatory efficacy of nsEP compared to longer pulses makes them advantageous for medical applications where the neuromuscular excitation is an unwanted side effect, such as electroporation-based cancer and tissue ablation.
Topics: Animals; Cell Line; Cell Membrane Permeability; Electric Stimulation; Electroporation; Humans; Membrane Potentials
PubMed: 32711366
DOI: 10.1016/j.bioelechem.2020.107598