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Bioelectrochemistry (Amsterdam,... Dec 2021The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological...
The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid.
Topics: Animals; CHO Cells; Cell Membrane; Cricetulus; Electroporation; Membrane Potentials; Photography; Single-Cell Analysis
PubMed: 34438186
DOI: 10.1016/j.bioelechem.2021.107929 -
International Journal of Molecular... Aug 2021The two-pore domain K subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all... (Review)
Review
The two-pore domain K subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K channels, and focus on the physiological roles of mechanically induced hyperpolarization.
Topics: Animals; Cell Membrane; Humans; Lipid Bilayers; Membrane Potentials; Physical Phenomena; Potassium Channels, Tandem Pore Domain
PubMed: 34445768
DOI: 10.3390/ijms22169062 -
ENeuro 2021Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons...
Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO/H-activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope hybridization, transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na and Ca currents previously associated with this distinctive electroresponsive property.
Topics: Action Potentials; Animals; Biological Clocks; Calcium Channels, L-Type; Central Pattern Generators; Membrane Potentials; Mice; Mice, Inbred CBA; Neurons; TRPM Cation Channels
PubMed: 34732535
DOI: 10.1523/ENEURO.0212-21.2021 -
International Journal of Molecular... Feb 2022The concerted function of the large number of ion channels expressed in excitable cells, including brain neurons, shapes diverse signaling events by controlling the... (Review)
Review
The concerted function of the large number of ion channels expressed in excitable cells, including brain neurons, shapes diverse signaling events by controlling the electrical properties of membranes. It has long been recognized that specific groups of ion channels are functionally coupled in mediating ionic fluxes that impact membrane potential, and that these changes in membrane potential impact ion channel gating. Recent studies have identified distinct sets of ion channels that can also physically and functionally associate to regulate the function of either ion channel partner beyond that afforded by changes in membrane potential alone. Here, we review canonical examples of such ion channel partnerships, in which a Ca channel is partnered with a Ca-activated K channel to provide a dedicated route for efficient coupling of Ca influx to K channel activation. We also highlight examples of non-canonical ion channel partnerships between Ca channels and voltage-gated K channels that are not intrinsically Ca sensitive, but whose partnership nonetheless yields enhanced regulation of one or the other ion channel partner. We also discuss how these ion channel partnerships can be shaped by the subcellular compartments in which they are found and provide perspectives on how recent advances in techniques to identify proteins in close proximity to one another in native cells may lead to an expanded knowledge of other ion channel partnerships.
Topics: Animals; Calcium; Ion Channel Gating; Ion Channels; Membrane Potentials; Neurons; Potassium; Signal Transduction
PubMed: 35216068
DOI: 10.3390/ijms23041953 -
Cells Jun 2020An improved understanding of fundamental physiological principles and progressive pathophysiological processes in human articular joints (e.g., shoulders, knees, elbows)... (Review)
Review
An improved understanding of fundamental physiological principles and progressive pathophysiological processes in human articular joints (e.g., shoulders, knees, elbows) requires detailed investigations of two principal cell types: synovial fibroblasts and chondrocytes. Our studies, done in the past 8-10 years, have used electrophysiological, Ca imaging, single molecule monitoring, immunocytochemical, and molecular methods to investigate regulation of the resting membrane potential (E) and intracellular Ca levels in human chondrocytes maintained in 2-D culture. Insights from these published papers are as follows: (1) Chondrocyte preparations express a number of different ion channels that can regulate their E. (2) Understanding the basis for E requires knowledge of a) the presence or absence of ligand (ATP/histamine) stimulation and b) the extraordinary ionic composition and ionic strength of synovial fluid. (3) In our chondrocyte preparations, at least two types of Ca-activated K channels are expressed and can significantly hyperpolarize E. (4) Accounting for changes in E can provide insights into the functional roles of the ligand-dependent Ca influx through store-operated Ca channels. Some of the findings are illustrated in this review. Our summary diagram suggests that, in chondrocytes, the K and Ca channels are linked in a positive feedback loop that can augment Ca influx and therefore regulate lubricant and cytokine secretion and gene transcription.
Topics: Animals; Calcium; Chondrocytes; Humans; Membrane Potentials; Potassium; Synovial Fluid
PubMed: 32610485
DOI: 10.3390/cells9071577 -
Antimicrobial Agents and Chemotherapy Jan 2019Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action...
Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in cells using fluorescence methods [PI and DiSC(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.
Topics: Anti-Bacterial Agents; Bacillus subtilis; Biological Transport; Cell Membrane Permeability; Daptomycin; Membrane Potentials; Microbial Sensitivity Tests; Pore Forming Cytotoxic Proteins
PubMed: 30323037
DOI: 10.1128/AAC.01589-18 -
Proceedings of the National Academy of... Apr 2024Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators...
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.
Topics: Membrane Potentials; Fluorescent Dyes; Action Potentials; Cell Membrane; Microscopy, Fluorescence
PubMed: 38551837
DOI: 10.1073/pnas.2315264121 -
PLoS Computational Biology Apr 2023When a neuron is driven beyond its threshold, it spikes. The fact that it does not communicate its continuous membrane potential is usually seen as a computational...
When a neuron is driven beyond its threshold, it spikes. The fact that it does not communicate its continuous membrane potential is usually seen as a computational liability. Here we show that this spiking mechanism allows neurons to produce an unbiased estimate of their causal influence, and a way of approximating gradient descent-based learning. Importantly, neither activity of upstream neurons, which act as confounders, nor downstream non-linearities bias the results. We show how spiking enables neurons to solve causal estimation problems and that local plasticity can approximate gradient descent using spike discontinuity learning.
Topics: Learning; Neurons; Membrane Potentials; Action Potentials; Models, Neurological
PubMed: 37014913
DOI: 10.1371/journal.pcbi.1011005 -
PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers.Journal of the American College of... Oct 2016Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent...
BACKGROUND
Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA).
OBJECTIVES
After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs.
METHODS
LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials.
RESULTS
Flecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c). Resting membrane potential was more depolarized in Pitx2c atria, and TWIK-related acid-sensitive K channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide's effects between wild-type and Pitx2c atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Na1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized.
CONCLUSIONS
PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.
Topics: Adult; Aged; Animals; Anti-Arrhythmia Agents; Atrial Fibrillation; Electrophysiological Phenomena; Female; Flecainide; Gene Expression Regulation; Heart Atria; Homeodomain Proteins; Humans; Male; Membrane Potentials; Mice; Middle Aged; Transcription Factors; Voltage-Gated Sodium Channel Blockers; Homeobox Protein PITX2
PubMed: 27765191
DOI: 10.1016/j.jacc.2016.07.766 -
Progress in Biophysics and Molecular... Nov 2021Hyperpolarization-activated cyclic nucleotide gated (HCN) channels and the current they carry, I, are widely and diversely distributed in the central nervous system... (Review)
Review
Hyperpolarization-activated cyclic nucleotide gated (HCN) channels and the current they carry, I, are widely and diversely distributed in the central nervous system (CNS). The distribution of the four subunits of HCN channels is variable within the CNS, within brain regions, and often within subcellular compartments. The precise function of I can depend heavily on what other channels are co-expressed. In this review, we give an overview of HCN channel structure, distribution, and modulation by cyclic adenosine monophosphate (cAMP). We then discuss HCN channel and I functions, where we have parsed the roles into two main effects: a steady effect on maintaining the resting membrane potential at relatively depolarized values, and slow channel dynamics. Within this framework, we discuss I involvement in resonance, synaptic integration, transmitter release, plasticity, and point out a special case, where the effects of I on the membrane potential and its slow channel dynamics have dual roles in thalamic neurons.
Topics: Cyclic Nucleotide-Gated Cation Channels; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Membrane Potentials; Neurons; Synapses
PubMed: 34181891
DOI: 10.1016/j.pbiomolbio.2021.06.002