A New Turgor/Membrane Potential Probe Simultaneously Measures Turgor and Electrical Membrane Potential

Abstract: A new combined turgor/membrane potential probe (T-EP probe) monitored cell turgor and membrane potential simultaneously in single giant cells. The new probe consisted of a silicone oil-filled micropipette (oil-microelectrode), which conducted electric current. Measurements of turgor and hydraulic conductivity were performed as with the conventional cell pressure probe besides the membrane potential. In internodal cells of Chara corallina, steady state turgor (0.5-0.7 MPa) and resting potentials (-200 to ?220 mV) in APW, and hydraulic conductivity (0.07 to 0.21 × 10～⁵ m s^?1 MPa^?1) were measured with the new probe, and cells exhibited healthy cytoplasmic streaming for at least 24 h during measurements. When internodal cells of Chara corallina were treated with 30, 20, 10, and 5 mM KCI, turgor responded immediately to all concentrations, and the osmotic changes in the medium were measured. Action potentials, which brought the membrane potential to a steady depolarization that measured the concentration difference of K⁺ in the medium, were induced in a concentration — dependent delay and occurred only 30, 20, and 10 mM of KCl. When the solution was changed back to APW, the repolarization of membrane potential consisted of a quick and a following slow phase. During the quick phase, which took place immediately and lasted 1 to 3 min, the plasma membrane remained activated. The membrane was gradually deactivated in the slow phase, and entirely deactivated when the membrane potential recovered to the resting potential in APW. Although the activated plasma membrane was permeable to K⁺, no major ion channels were activated on the tonoplast, and therefore, internodal cells of Chara corallina did not regulate turgor when osmotic potential changed in the surrounding medium.     

The Membrane Potential of Nitella translucens

The effects of changing the external concentrations of Na, K,Ca, and Cl on the potentials of the cytoplasm and the vacuolewith respect to the bathing medium of the internodal cells ofNitella translucens have been investigated. The potential differencebetween the vacuole and the cytoplasm is practically unaffectedby the concentration changes. The observed changes of potentialdifference are therefore attributed to the boundary separatingthe cytoplasm from the medium; this boundary is possibly a plasmalemma–cellwall complex. The difference of potential between the cell walland the medium has also been measured and, in the presence ofCa, shown to be markedly sensitive only to the external Ca concentration.The results are divided into two sections: (a) for cells pretreatedin 5 mM NaCl, the subsequent experiments being carried out inCa-free media, and (b) for cells initially immersed in a standardartificial pond water containing the chlorides of Na, K, Ca.With the pretreated cells the external Na/K ratio was variedwith the total NaCl+KCl concentration kept constant at 1.1 mM.The results suggest that over a limited range of concentrationsthe cytoplasm-medium potential difference can be described byan equation similar in form to a Goldman equation but containingonly terms for Na and K, the average value of the permeabilityratio (= P_Na/P_K) being 0.27. In the presence of Ca the effectsof Na and K on the cytoplasm-medium potential difference aregreatly reduced, while the effect of Ca is relatively large.The results cannot be fitted to any form of Goldman equationcontaining terms for the major ions. The possibility of a contributionto the plasmalemma potential from electrogenic pumps is brieflydiscussed. Measurements of the Na and K content of the cytoplasmand the vacuole have been made for the pretreated cells. TheNa concentration in the cytoplasm is 37 mM and in the vacuole73 mM; the K concentration is 93 mM in the cytoplasm and 67mM in the vacuole. The Nernst potentials for both ions are comparedwith the cytoplasm-medium and cytoplasm-vacuole potential differences.This analysis shows that Na is actively transported from thecytoplasm into the medium as well as into the Vacuole; K ispumped into the cytoplasm from the medium but appears to beclose to electrochemical equilibrium across the tonoplast. ThisConfirms previously published work.     

Membrane Potential of the Root-base segment

By cutting segments of different lengths from 1-week-old barleyroots and interposing the remainder as a membrane in concentrationcells, it was found that the root-base segment of 2—3cm is similar to a homogeneous membrane with constant transportnumbers for ions. The transport number for potassium was foundto be 0.52 indicating that the root-base segment possesses electricfields of low intensities with little effect on the mobilitiesof the ions. The effect of pH on the potentials and the pointof zero charge of the root-base segment show close similaritiesto earlier results obtained with whole roots.     

The Membrane Potential of Acetabularia mediterranea

The cytoplasm of an Acetabularia cell is normally at a potential of about -170 mv relative to the external solution; the vacuole is also at this potential. Although there is strict flux equilibrium for all ions, the potential is more negative than the Nernst potentials of any of the permeating ions. Darkness, CCCP, low temperature, and reducing [Cl^-]_o by a factor of 25 all rapidly depolarize the membrane and inhibit Cl^- influx. Some of these treatments do not inhibit the effluxes of K⁺ and Na⁺. Increasing [K⁺]_o also depolarizes the membrane both under normal conditions and at low temperature; in the latter case the membrane is partially depolarized in normal seawater (low [K⁺]_o) and in high [K⁺]_o positive potentials of up to +15 mv are attained. It is concluded that the membrane potential is controlled by the electrogenic influx of Cl^-, and also, at least in some circumstances, by the diffusion of K⁺. In addition, it is suggested that electrogenic efflux of H⁺ may be important in transient nonequilibrium situations. An Appendix deals with the interpretation of simple nonsteady-state tracer kinetic data.     

Evaluating Mitochondrial Membrane Potential in Cells

Permeant cationic fluorescent probes are widely employed to monitor mitochondrial transmembrane potential and its changes. The application of such potential-dependent probes in conjunction with both fluorescence microscopy and fluorescence spectroscopy allows the monitoring of mitochondrial membrane potential in individual living cells as well as in large population of cells. These approaches to the analysis of membrane potential is of extremely high value to obtain insights into both the basic energy metabolism and its dysfunction in pathologic cells. However, the use of fluorescent molecules to probe biological phenomena must follow the awareness of some principles of fluorescence emission, quenching, and quantum yield since it is a very sensitive tool, but because of this extremely high sensitivity it is also strongly affected by the environment. In addition, the instruments used to monitor fluorescence and its changes in biological systems have also to be employed with cautions due to technical limits that may affect the signals. We have therefore undertaken to review the most currently used analytical methods, providing a summary of practical tips that should precede data acquisition and subsequent analysis. Furthermore, we discuss the application and feasibility of various techniques and discuss their respective strength and weakness.     

Functional Significance of the Mitochondrial Membrane Potential

The electrical polarization of the inner mitochondrial membrane largely determines the electrochemical potential of hydrogen ifons, being thereby a significant factor in the energy transformation during oxidation of respiratory substrates and its accumulation in the form of newly synthesized ATP. However, the gradient of the electric potential on the inner mitochondrial membrane (ΔΨm) performs a number of functions not related to energy production. Even under hypoxic conditions, precluding the formation of ATP in mitochondria through oxidative phosphorylation, mitochondria maintain their ΔΨm at the expense of the hydrolysis of cellular ATP, which indicates the exceptional importance of ΔΨm for non-energetic functions of mitochondria. Among these functions, the mitochondrial inward transport of metal cations and proteins carrying a positively charged amino acid sequence and export of anions including nucleic acids possibly providing retrograde signaling, seem very important and essential for maintaining mitochondrial structure and metabolism. ΔΨm is a powerful regulator of mitochondrial generation of reactive oxygen species that perform physiological and pathological functions. And finally, ΔΨm is a critical element in the mechanism of disposal of dysfunctional mitochondria, the so-called quality control machinery of mitochondria. The disturbance of this mechanism leads to increase of heterogeneity in the population of mitochondria in the cell, and the degree of heterogeneity can be considered as an indicator of the pathological cellular phenotype. Correlation between Ψm and cell functions is difficult to identify without adequate quantitative estimates of the magnitude of ΔΨm, which are complicated due to several cellular and mitochondrial processes that affect the experimentally obtained values. Recommendations for assessing the contribution of these processes and avoiding artifacts in the measurements of ΔΨm by standard methods are given.     

A Practical Implicit Membrane Potential for NMR Structure Calculations of Membrane Proteins

The highly anisotropic environment of the lipid bilayer membrane imposes significant constraints on the structures and functions of membrane proteins. However, NMR structure calculations typically use a simple repulsive potential that neglects the effects of solvation and electrostatics, because explicit atomic representation of the solvent and lipid molecules is computationally expensive and impractical for routine NMR-restrained calculations that start from completely extended polypeptide templates. Here, we describe the extension of a previously described implicit solvation potential, eefxPot, to include a membrane model for NMR-restrained calculations of membrane protein structures in XPLOR-NIH. The key components of eefxPot are an energy term for solvation free energy that works together with other nonbonded energy functions, a dedicated force field for conformational and nonbonded protein interaction parameters, and a membrane function that modulates the solvation free energy and dielectric screening as a function of the atomic distance from the membrane center, relative to the membrane thickness. Initial results obtained for membrane proteins with structures determined experimentally in lipid bilayer membranes show that eefxPot affords significant improvements in structural quality, accuracy, and precision. Calculations with eefxPot are straightforward to implement and can be used to both fold and refine structures, as well as to run unrestrained molecular-dynamics simulations. The potential is entirely compatible with the full range of experimental restraints measured by various techniques. Overall, it provides a useful and practical way to calculate membrane protein structures in a physically realistic environment.     

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.     

Cell Surface Deformation during an Action Potential

The excitation of many cells and tissues is associated with cell mechanical changes. The evidence presented herein corroborates that single cells deform during an action potential. It is demonstrated that excitation of plant cells (Chara braunii internodes) is accompanied by out-of-plane displacements of the cell surface in the micrometer range (～1–10 μm). The onset of cellular deformation coincides with the depolarization phase of the action potential. The mechanical pulse: 1) propagates with the same velocity as the electrical pulse (within experimental accuracy, ～10 mm s^?1), 2) is reversible, 3) in most cases is of biphasic nature (109 out of 152 experiments), and 4) is presumably independent of actin-myosin-motility. The existence of transient mechanical changes in the cell cortex is confirmed by micropipette aspiration experiments. A theoretical analysis demonstrates that this observation can be explained by a reversible change in the mechanical properties of the cell surface (transmembrane pressure, surface tension, and bending rigidity). Taken together, these findings contribute to the ongoing debate about the physical nature of cellular excitability.     

Membrane Potential Measurements in Cultured Intestinal Villi

Fragmented epithelia of newborn rat small intestine were successfully cultured for periods of up to 4 weeks. Stable intracellular recordings of membrane potential were obtained from these cultured cells. Membrane resting potential varied according to cell location along a villus. The potentials ranged from -70 to -15 mV, being highest at the tip of the villus. The mean resting potential and membrane resistance were -72.4 mV and 8.6 M Ω, respectively. The membrane potential was markedly dependent on the extracellular K⁺ concentration ([K]₀], but not significantly on [Na]₀ and [Cl]₀-Deprivation of Ca²⁺ from the surrounding medium depolarized the membrane by 20 mV. When the cells were cooled down to 6°C, membrane potential was reduced by 40 mV. Based on these data, basic mechanisms underlying the resting potential are discussed in connection with cell differentiation or maturation.     

用电压敏感染料光学记录膜电位

应用传统电生理方法如微电极和膜片钳技术,在记录较小的神经细胞和纤细的神经突起膜电位及同步记录神经细胞群的电活动等方面目前仍是一大难题。随着生理科学和神经生物学的发展,利用电压敏感染料光学记录膜电位技术已成为一种较为理想的新手段。本文对光学记录膜电位技术的发展史、染料特性和作用机制、光学成像及膜电位记录原理、目前的光学方法中某些不足及未来前景等做了较系统的介绍,并且简述了光学记录膜电位在电生理和神经生物学中的应用。     

Visualization of Dynamic Sub-microsecond Changes in Membrane Potential

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ～25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.     

Chloride Conductance Determining Membrane Potential of Rabbit Articular Chondrocytes

Membrane conductance of cultured rabbit articular chondrocytes was characterized by means of the patch-clamp technique. The resting membrane potential of the articular chondrocytes was about -42 mV. The membrane potential shifted in accordance with the prediction by the Nernst equation for Cl- when intracellular and extracellular concentrations of Cl- were changed. On the other hand, change in extracellular concentration of K+ produced no shift in the membrane potential of chondrocytes. The Cl- channel blocker 4-acetamido-4'-isothiocyanatostilbene-2'2-disulfonic acid (SITS) depolarized the membrane potential. These findings suggest that the membrane potential of the chondrocytes is determined mainly by Cl- conductance. Using the cell-attached patch-clamp method, a large unitary conductance of 217 pS was observed in the articular chondrocytes. The unitary current was reversibly blocked by SITS. Therefore, the unitary current was carried by Cl-. The Cl- channel showed voltage-dependent activation and the channels exhibited long-lasting openings. Therefore, the membrane potential of rabbit cultured articular chondrocytes was mainly determined by the activities of the large-conductance and voltage-dependent Cl- channels.     

Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research

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Functionalized Amphipols: A Versatile Toolbox Suitable for Applications of Membrane Proteins in Synthetic Biology

Amphipols are amphipathic polymers that stabilize membrane proteins isolated from their native membrane. They have been functionalized with various chemical groups in the past years for protein labeling and protein immobilization. This large toolbox of functionalized amphipols combined with their interesting physico-chemical properties give opportunities to selectively add multiple functionalities to membrane proteins and to tune them according to the needs. This unique combination of properties makes them one of the most versatile strategies available today for exploiting membrane proteins onto surfaces for various applications in synthetic biology. This review summarizes the properties of functionalized amphipols suitable for synthetic biology approaches.     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)