Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment

The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation.     

Microscopic description of voltage effects on ion-driven cotransport systems

A microscopic model for the analysis of voltage effects on ion-driven cotransport systems is described. The model is based on the notion that the voltage dependence of a given rate constant is directly related to the amount of charge which is translocated in the corresponding reaction step. Charge translocation may result from the movement of an ion along the transport pathway, from the displacement of charged ligand groups of the ion-binding site, or from reorientation of polar residues of the protein in the course of a conformational transition. The voltage dependence of overall transport rate is described by a set of dimensionless coefficients reflecting the dielectric distances over which charge is displaced in the elementary reaction steps. The dielectric coefficients may be evaluated from the shape of the experimental flux-voltage curve if sufficient information on the rate constants of the reaction cycle is available. Examples of flux-voltage curves which are obtained by numerical simulation of the transport model are given for a number of limiting cases.     

Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.     

On the mechanics of viscous bodies and elongation in ellipsoidal cells

After deriving some auxiliary equations for the average elongation of a viscous body under the action of forces derived from a potential, the diffusion problem for an ellipsoidal cell with a constant rate of reaction is solved for the case of an infinite permeability. The equation of elongation of such a cell under the influence of diffusion forces is derived, and compared with the, approximate expression obtained by N. Rashevsky for any kind of oblong cell. The two equations are in fair agreement. Effects of constant and variable surface tension are studied.     

ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.

ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.     

Stereological analysis of the guinea pig pancreas. I. Analytical model and quantitative description of nonstimulated pancreatic exocrine cells

A stereological model which provides detailed quantitative information on the structure of the fasted, nonstimulated gland has been developed for the guinea pig pancreas. The model consists of morphologically defined space and membrane compartments which were used to describe the general composition of the tissue and the specific components of exocrine cells. The results are presented, where appropriate, relative to a cubic centimeter of pancreas, a cubic centimeter of exocrine cell cytoplasm, and to the volume of an average exocrine cell. The exocrine cells, accounting for 82% of the pancreas volume, consisted of 54% cytoplasmic matrix, 22% rough-surfaced endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules, and 0.7% condensing vacuoles. Their total membrane surface area was distributed as follows: 60% RER, 21% mitochondria, 9.9% Golgi apparatus, 4.8% plasma membranes, 2.6% zymogen granules, 1.8% plasma membrane vesicles, and 0.4% condensing vacuoles. The application of this model to the study of membrane movements associated with the secretory process is discussed within the framework of an analytical approach.     

Formation of the transmembrane electrochemical potential on Staphylococcus cells and membrane vesicles

The generation of transmembrane difference of electrochemical potentials was registered on the intact cells and ultrasonication-obtained membrane vesicles of Staphylococcus aureus with the application of transmembrane electrophoresis of permeant anions, potassium transport in the presence of valinomycin and 8-anilinonaphthalene-1-sulphonate fluorescence. The membrane potential is formed when the chain of electron transfer or H+-ATPase functions or when the pH gradient varies (the nonenzymic pathway). M-chlorinecarbonylcyanidephenylhydrazonium, a protonophore uncoupler potassium cyanide, an inhibitor of the respiratory chain, N',N-dicyclohexylcarbodiimide, an inhibitor of ATPase, cause the membrane potential dissipation. The orientation of the transmembrane electric field is as follows: "minus" inside cells and "plus" inside membrane vesicles.     

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane

The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2±0.2kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9?. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence.     

The transmembrane domain of surfactant protein C precursor determines the morphology of the induced membrane compartment in CHO cells

Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.     

The relationship between sensitivity to 60-Hz electric fields and induced transmembrane potentials in plants root cells

Summary Growth rates and cell diameters were determined from 12 species of plant roots exposed to a 60-Hertz (Hz) electric field of 360 Volts per meter (V/m) in an aqueous inorganic nutrient medium [conductivity: 0.07–0.09 Siemens per meter (S/m)]. The degree of growth depression ranged from zero to nearly 100 percent of control. Cell diameters ranged from 13.5 to 31.8 µm as an averaged value for procambial, cortical, and meristem cells. Sensitivity to the electric field as determined by root growth rate reduction increased with increasing cell size. Sensitivity also increased with increase in 60 Hz induced transmembrane potentials; the transmembrane potential threshold for growth reduction was about 6.0 mV and the potential for near-complete cessation of growth was about 10–11 mV.Two different hypothetical mechanisms of action by which applied electric fields induce biological effects at the cellular level were tested. The two mechanisms pertain to different possible modes of action of applied electric fields: one mechanism postulates the involvement of the transmembrane field, the other mechanism postulates the tangential electric field as the important factor for inducing biological effects. The data support the transmembrane and not the tangential field mechanism. It is concluded that the effects observed are consistent with a membrane related mechanism and that there is a narrow range (a few mV) between threshold and debilitating induced membrane potentials.     

Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC

Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 a helix and 12 strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel (s). This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.This work was supported by grants from the Office of Naval Research (N00014-90-J-1024) and the National Institutes of Health (GM 35759). Present address: Department of Physiology, 6811 Med. Sciences Bldg 2, University of Michigan, Ann Arbor, MI 48109 Present address: Department of Physiology, K.U. Leuven Medical School, Gasthuijsberg, 3000 Leuven, Belgium     

Methods of measuring the transmembrane electrical potential in cells

The methods of intracellular microelectrodes, penetrating ions and potential-sensitive fluorescent probes are considered for their possibility to be used for quantitative estimation of transmembrane electrical potentials (TMP) of small cells (mainly through the example of lymphocytes). The following fluorescent methods are described in detail: separate measurement of two TMP components--potentials on the plasma and mitochondrial membranes of a cell; recording of individual differences of cells according to the TMP value. It is supposed that heterogeneity of cells by the TMP value (in particular, the presence of depolarized cells) may be responsible for errors and divergences of the TMP mean values measured by different methods.     

The theory of the frequency response of ellipsoidal biological cells in rotating electrical fields

In this paper we have presented in as compact a form as possible the theoretical formalism that is needed to predict the frequency response of a biological cell of arbitrary ellipsoidal shape to a frequency dependant rotating external field. The formalism is much more complicated than that for a spherical or cylindrical cell where the radial vector is always parallel to the surface normal at each point of the surface. In addition to providing the theory we have demonstrated that the spin rate and its frequency dependance is very intimately related to the electrical properties of the cell interior and to that of the suspending fluid. It is possible to probe these properties of the cell and its environment by utilizing this technique. This aspect has been demonstrated by examining rotational changes as a function of the conductivity of both the cell interior and its suspending liquid. We also have shown, by considering a very simple model for the cell and the two dielectric constants, that the frequency spectrum is shape dependant. All our calculations have been carried out for "lossy" systems with frictional dissipation where energy minimization methods are no longer applicable. The invariant form of the Poynting vector forms the basis of the method.