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International Journal of Molecular... Aug 2023The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal...
A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development.
The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H fluxes with a permeability ratio of P/P = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K and H conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.
Topics: High-Throughput Screening Assays; Membrane Potentials; Cardiac Electrophysiology; Cations; Lysosomes
PubMed: 37628970
DOI: 10.3390/ijms241612788 -
Journal of Biological Physics Dec 2023The membrane potential of a cell (V) regulates several physiological processes. The voltage sensor domain (VSD) is a region that confers voltage sensitivity to different... (Review)
Review
The membrane potential of a cell (V) regulates several physiological processes. The voltage sensor domain (VSD) is a region that confers voltage sensitivity to different types of transmembrane proteins such as the following: voltage-gated ion channels, the voltage-sensing phosphatase (Ci-VSP), and the sperm-specific Na/H exchanger (sNHE). VSDs contain four transmembrane segments (S1-S4) and several positively charged amino acids in S4, which are essential for the voltage sensitivity of the protein. Generally, in response to changes of the V, the positive residues of S4 displace along the plasma membrane without generating ionic currents through this domain. However, some native (e.g., Hv1 channel) and mutants of VSDs produce ionic currents. These gating pore currents are usually observed in VSDs that lack one or more of the conserved positively charged amino acids in S4. The gating pore currents can also be induced by the isolation of a VSD from the rest of the protein domains. In this review, we summarize gating pore currents from all families of proteins with VSDs with classification into three cases: (1) pathological, (2) physiological, and (3) artificial currents. We reinforce the model in which the position of S4 that lacks the positively charged amino acid determines the voltage dependency of the gating pore current of all VSDs independent of protein families.
Topics: Male; Humans; Ion Channel Gating; Semen; Protein Domains; Membrane Potentials; Amino Acids
PubMed: 37851173
DOI: 10.1007/s10867-023-09645-z -
Biomedical Engineering Online Jan 2024Bioelectric signals, whether exogenous or endogenous, play crucial roles in the life processes of organisms. Recently, the significance of bioelectricity in the field of... (Review)
Review
BACKGROUND
Bioelectric signals, whether exogenous or endogenous, play crucial roles in the life processes of organisms. Recently, the significance of bioelectricity in the field of dentistry is steadily gaining greater attention.
OBJECTIVE
This narrative review aims to comprehensively outline the theory, physiological effects, and practical applications of bioelectricity in dental medicine and to offer insights into its potential future direction. It attempts to provide dental clinicians and researchers with an electrophysiological perspective to enhance their clinical practice or fundamental research endeavors.
METHODS
An online computer search for relevant literature was performed in PubMed, Web of Science and Cochrane Library, with the keywords "bioelectricity, endogenous electric signal, electric stimulation, dental medicine."
RESULTS
Eventually, 288 documents were included for review. The variance in ion concentration between the interior and exterior of the cell membrane, referred to as transmembrane potential, forms the fundamental basis of bioelectricity. Transmembrane potential has been established as an essential regulator of intercellular communication, mechanotransduction, migration, proliferation, and immune responses. Thus, exogenous electric stimulation can significantly alter cellular action by affecting transmembrane potential. In the field of dental medicine, electric stimulation has proven useful for assessing pulp condition, locating root apices, improving the properties of dental biomaterials, expediting orthodontic tooth movement, facilitating implant osteointegration, addressing maxillofacial malignancies, and managing neuromuscular dysfunction. Furthermore, the reprogramming of bioelectric signals holds promise as a means to guide organism development and intervene in disease processes. Besides, the development of high-throughput electrophysiological tools will be imperative for identifying ion channel targets and precisely modulating bioelectricity in the future.
CONCLUSIONS
Bioelectricity has found application in various concepts of dental medicine but large-scale, standardized, randomized controlled clinical trials are still necessary in the future. In addition, the precise, repeatable and predictable measurement and modulation methods of bioelectric signal patterns are essential research direction.
Topics: Cell Membrane; Electrophysiological Phenomena; Ion Channels; Mechanotransduction, Cellular; Membrane Potentials
PubMed: 38172866
DOI: 10.1186/s12938-023-01189-6 -
PloS One 2023Living organisms maintain a resting membrane potential, which plays an important role in various biophysical and biological processes. In the context of medical...
Living organisms maintain a resting membrane potential, which plays an important role in various biophysical and biological processes. In the context of medical applications, irreversible electroporation (IRE) is a non-thermal and minimally invasive technique that utilizes precisely controlled electric field pulses of micro- to millisecond durations to effectively ablate cancer and tumor cells. Previous studies on IRE-induced rupture of cell-mimetic giant unilamellar vesicles (GUVs) have primarily been conducted in the absence of membrane potentials. In this study, we investigated the electroporation of GUVs, including parameters such as the rate constant of rupture and the probability of rupture, in the presence of various negative membrane potentials. The membranes of GUVs were prepared using lipids and channel forming proteins. As the membrane potential increased from 0 to -90 mV, the rate constant of rupture showed a significant increase from (7.5 ± 1.6)×10-3 to (35.6 ± 5.5)×10-3 s-1. The corresponding probability of rupture also exhibited a notable increase from 0.40 ± 0.05 to 0.68 ± 0.05. To estimate the pore edge tension, the electric tension-dependent logarithm of the rate constant was fitted with the Arrhenius equation for different membrane potentials. The presence of membrane potential did not lead to any significant changes in the pore edge tension. The increase in electroporation is reasonably explained by the decrease in the prepore free energy barrier. The choice of buffer used in GUVs can significantly influence the kinetics of electroporation. This study provides valuable insights that can contribute to the application of electroporation techniques in the biomedical field.
Topics: Membrane Potentials; Unilamellar Liposomes; Electroporation; Electroporation Therapies; Biophysics
PubMed: 37699026
DOI: 10.1371/journal.pone.0291496 -
Redox Report : Communications in Free... Dec 2023Melittin, a naturally occurring polypeptide found in bee venom, has been recognized for its potential anti-tumor effects, particularly in the context of lung cancer. Our...
Melittin, a naturally occurring polypeptide found in bee venom, has been recognized for its potential anti-tumor effects, particularly in the context of lung cancer. Our previous study focused on its impact on human lung adenocarcinoma cells A549, revealing that melittin induces intracellular reactive oxygen species (ROS) burst and oxidative damage, resulting in cell death. Considering the significant role of mitochondria in maintaining intracellular redox levels and ROS, we further examined the involvement of mitochondrial damage in melittin-induced apoptosis in lung cancer cells. Our findings demonstrated that melittin caused changes in mitochondrial membrane potential (MMP), triggered mitochondrial ROS burst (Figure 1), and activated the mitochondria-related apoptosis pathway Bax/Bcl-2 by directly targeting mitochondria in A549 cells (Figure 2). Further, we infected A549 cells using a lentivirus that can express melittin-Myc and confirmed that melittin can directly target binding to mitochondria, causing the biological effects described above (Figure 2). Notably, melittin induced mitochondrial damage while inhibiting autophagy, resulting in abnormal degradation of damaged mitochondria (Figure 5). To summarize, our study unveils that melittin targets mitochondria, causing mitochondrial damage, and inhibits the autophagy-lysosomal degradation pathway. This process triggers mitoROS burst and ultimately activates the mitochondria-associated Bax/Bcl-2 apoptotic signaling pathways in A549 cells.
Topics: Humans; Mitophagy; A549 Cells; Melitten; Reactive Oxygen Species; bcl-2-Associated X Protein; Mitochondria; Apoptosis; Membrane Potential, Mitochondrial; Lung Neoplasms
PubMed: 38041592
DOI: 10.1080/13510002.2023.2284517 -
Nature Communications Oct 2023Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated...
Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.
Topics: Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Membrane Lipids; Ion Channel Gating; Membrane Potentials; Cyclic Nucleotide-Gated Cation Channels
PubMed: 37852983
DOI: 10.1038/s41467-023-42363-7 -
Advances in Physiology Education Sep 2023A task force of physiology educators from 25 Australian universities generated an Australia-wide consensus on seven core concepts for physiology curricula. One adopted...
A task force of physiology educators from 25 Australian universities generated an Australia-wide consensus on seven core concepts for physiology curricula. One adopted core concept was "cell membrane," defined as "Cell membranes determine what substances enter or leave the cell and its organelles. They are essential for cell signaling, transport, and other cellular functions." This concept was unpacked by a team of 3 Australian physiology educators into 4 themes and 33 subthemes arranged in a hierarchical structure up to 5 levels deep. The four themes related to defining the cell membrane, cell membrane structure, transport across cell membranes, and cell membrane potentials. Subsequently, 22 physiology educators with a broad range of teaching experience reviewed and assessed the 37 themes and subthemes for importance for students to understand and the level of difficulty for students on a 5-point Likert scale. The majority (28) of items evaluated were rated as either Essential or Important. was rated as less important than the other three themes. was rated most difficult, while was rated as the easiest. The importance of cell membranes as a key aspect of biomedical education received strong support from Australian educators. The unpacking of the themes and subthemes within the cell membrane core concept provides guidance in the development of curricula and should facilitate better identification of the more challenging aspects within this core concept and help inform the time and resources required to support student learning. The "cell membrane" core concept was unpacked by a team of Australian physiology educators into a conceptual framework to provide guidance for students and educators. Key themes in the cell membrane core concept were cell membrane definition and structure, transport across cell membranes, and membrane potentials. Australian educators reviewing the framework identified cell membrane as an essential yet relatively simple core concept, suggesting that this is well-placed in foundational physiology courses across a diverse range of degrees.
Topics: Humans; Australia; Curriculum; Cell Membrane; Students; Universities; Physiology
PubMed: 37318997
DOI: 10.1152/advan.00143.2022 -
Proceedings of the National Academy of... Aug 2023Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed...
Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity. One dye was applied to ex vivo murine heart with excitation at 730 nm, by far the longest wavelength reported in voltage imaging. This VSD resolves cardiac action potentials in single trials with 12% ΔF/F per action potential. The well-separated excitation spectra between these long-wavelength VSDs and channelrhodopsin (ChR2) enabled monitoring of action potential propagation in ChR2 hearts without any perturbation of electrical dynamics. Importantly, by employing spatially localized optogenetic manipulation, action potential dynamics can be assessed in an all-optical fashion with no artifact related to optical cross-talk between the reporter and actuator. These new environmentally sensitive chromene-based chromophores are also likely to have applications outside voltage imaging.
Topics: Mice; Animals; Fluorescent Dyes; Action Potentials; Heart; Fluorescence
PubMed: 37579138
DOI: 10.1073/pnas.2305093120 -
Biochemistry. Biokhimiia Oct 2023Heme-copper respiratory oxidases are highly efficient molecular machines. These membrane enzymes catalyze the final step of cellular respiration in eukaryotes and many... (Review)
Review
Heme-copper respiratory oxidases are highly efficient molecular machines. These membrane enzymes catalyze the final step of cellular respiration in eukaryotes and many prokaryotes: the transfer of electrons from cytochromes or quinols to molecular oxygen and oxygen reduction to water. The free energy released in this redox reaction is converted by heme-copper respiratory oxidases into the transmembrane gradient of the electrochemical potential of hydrogen ions ). Heme-copper respiratory oxidases have a unique mechanism for generating H+, namely, a redox-coupled proton pump. A combination of direct electrometric method for measuring the kinetics of membrane potential generation with the methods of prestationary kinetics and site-directed mutagenesis in the studies of heme-copper oxidases allows to obtain a unique information on the translocation of protons inside the proteins in real time. The review summarizes the data of studies employing time-resolved electrometry to decipher the mechanisms of functioning of these important bioenergetic enzymes.
Topics: Electron Transport Complex IV; Heme; Membrane Potentials; Oxidoreductases; Oxidation-Reduction; Protons; Ceruloplasmin; Oxygen
PubMed: 38105021
DOI: 10.1134/S0006297923100085 -
International Journal of Molecular... Sep 2023This review analyzes the causes and consequences of apoptosis resulting from oxidative stress that occurs in mitochondria and cells exposed to the toxic effects of... (Review)
Review
This review analyzes the causes and consequences of apoptosis resulting from oxidative stress that occurs in mitochondria and cells exposed to the toxic effects of different-valence heavy metals (Ag, Tl, Hg, Cd, Pb, Al, Ga, In, As, Sb, Cr, and U). The problems of the relationship between the integration of these toxic metals into molecular mechanisms with the subsequent development of pathophysiological processes and the appearance of diseases caused by the accumulation of these metals in the body are also addressed in this review. Such apoptosis is characterized by a reduction in cell viability, the activation of caspase-3 and caspase-9, the expression of pro-apoptotic genes ( and ), and the activation of protein kinases (ERK, JNK, p53, and p38) by mitogens. Moreover, the oxidative stress manifests as the mitochondrial permeability transition pore (MPTP) opening, mitochondrial swelling, an increase in the production of reactive oxygen species (ROS) and HO, lipid peroxidation, cytochrome c release, a decline in the inner mitochondrial membrane potential (ΔΨ), a decrease in ATP synthesis, and reduced glutathione and oxygen consumption as well as cytoplasm and matrix calcium overload due to Ca release from the endoplasmic reticulum (ER). The apoptosis and respiratory dysfunction induced by these metals are discussed regarding their interaction with cellular and mitochondrial thiol groups and Fe metabolism disturbance. Similarities and differences in the toxic effects of Tl from those of other heavy metals under review are discussed. Similarities may be due to the increase in the cytoplasmic calcium concentration induced by Tl and these metals. One difference discussed is the failure to decrease Tl toxicity through metallothionein-dependent mechanisms. Another difference could be the decrease in reduced glutathione in the matrix due to the reversible oxidation of Tl to Tl near the centers of ROS generation in the respiratory chain. The latter may explain why thallium toxicity to humans turned out to be higher than the toxicity of mercury, lead, cadmium, copper, and zinc.
Topics: Humans; Reactive Oxygen Species; Calcium; Hydrogen Peroxide; Mitochondrial Membrane Transport Proteins; Mitochondria; Apoptosis; Oxidative Stress; Cadmium; Glutathione; Metals, Heavy; Membrane Potential, Mitochondrial
PubMed: 37833908
DOI: 10.3390/ijms241914459