Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

Role of membrane surface potential and other factors in the uptake of non-transferrin-bound iron by reticulocytes

Reticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non-transferrin-bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo-cytes incubated with ⁵⁹Fe-labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K⁺, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono-, di-, tri-, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley-Liss, Inc.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.     

Modifications in erythrocyte membrane zeta potential by Plasmodium falciparum infection

The zeta potential (ZP) is an electrochemical property of cell surfaces that is determined by the net electrical charge of molecules exposed at the surface of cell membranes. Membrane proteins contribute to the total net electrical charge of cell surfaces and can alter ZP through variation in their copy number and changes in their intermolecular interactions. Plasmodium falciparum extensively remodels its host red blood cell (RBC) membrane by placing 'knob'-like structures at the cell surface. Using an electrophoretic mobility assay, we found that the mean ZP of human RBCs was -15.7 mV. In RBCs infected with P. falciparum trophozoites ('iRBCs'), the mean ZP was significantly lower (-14.6 mV, p<0.001). Removal of sialic acid from the cell surface by neuraminidase treatment significantly decreased the ZP of both RBCs (-6.06 mV) and iRBCs (-4.64 mV). Parasite-induced changes in ZP varied by P. falciparum clone and the presence of knobs on the iRBC surface. Variations in ZP values were accompanied by altered binding of iRBCs to human microvascular endothelial cells (MVECs). These data suggest that parasite-derived knob proteins contribute to the ZP of iRBCs, and that electrostatic and hydrophobic interactions between iRBC and MVEC membranes are involved in cytoadherence.     

Fluorometric estimation of surface potential change associated with chemotactic stimulation in Tetrahymena pyriformis

For the quantitative estimation of surface potential change in intact cells a method was devised with the use of fluorescent probes, 8-anilino-1-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPN). Estimated values in liposomes were compared with changes in the zeta potential determined from electrophoresis. Both values agreed within the experimental variation, showing the usefulness of the method. The method was also applied to Tetrahymena pyriformis, which exhibits chemotaxis to various chemical stimuli. The surface potential change was observed when the cell was stimulated not only by inorganic salts but also by electrically neutral, hydrophobic compounds. The surface potential started to change in accordance with the depolarization of the membrane potential, except for the case of K⁺. Changes in the surface potential of T. pyriformis in response to Ca²⁺ and K⁺ were compared with those in the membrane potential. The quantitative contribution of the surface potential to cell depolarization associated with chemoreception is discussed.     

Dunaliella acidophila: an algae with a positive zeta potential at its optimal pH for growth*

Abstract. The zeta potential (which approximates the surface potential) of the acid resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) and the salt resistant D. parva (grown at pH 7.6) were calculated from the electrophoretic mobility of cells as determined by means of free-flow electrophoresis. Dunaliella acidophila cells exhibit a positive zeta potential (+5 to +20mV) at acidic external pH values, whereas negative zeta potentials (-30 mV) were measured at neutral pH values. Negative zeta potentials of the same order of magnitude were also measured for D. parva cells (pH 7.6). Low concentrations of La³⁺ and A1³⁺ did not affect the positive zeta potential of D. acidophila at acidic pH values, whereas higher concentrations caused a shift to more positive potentials. However, at neutral pH these cations caused a significant decrease of the negative zeta potential. The impermeant polycation poly-L-lysine acted in a similar manner to A1³⁺ or La³⁺. The effect of Impermeant cations and anions on various physiological reactions also supports the existence of a positive zeta potential for D. acidophila and of a negative zeta potential for D. parva: polycations such as DEAE-dextran and poly-L-lysine strongly inhibitied photosynthesis and mobility of D. parva, but did not affect these reactions in D. acidophila. Comparable differential inhibitions were also observed for A1³⁺ and La³⁺. Impermeant anions such as Dextran-sulfate exhibited effects in the opposite direction: inhibition was stronger with D. acidophila and weaker with D. parva. However, the differential inhibition by impermeant anions was much less pronounced in comparison with impermeant cations due to the relatively high pK_a values of anionic solutes and consequently relatively high protonation at pH 1.0. The physiological consequences of an asymmetrically charged plasma membrane (excess of positive charges outside, excess of negative charges on the cytoplasmic side) of D. acidophila are discussed in regard to the extreme acid resistance of this alga and its resistance to cationic toxic solutes in industrial wastes.     

Osmotic dependence of the transmembrane potential difference of broadbean mesocarp cells

Pod walls of broadbean (Vicia faba L. cv Aguadulce) were harvested at the import (S₁), at the transition (S₂) or at the export (S₃) phase for assimilate transport. Measurements of the transmembrane potential difference (PD) of mesocarp cells were made under various osmotic conditions. Internal osmotic potentials and cell turgor were calculated from osmolality measurements of cell saps recovered by freeze-thawing, after correction for the contribution of the free-space solution. Changes in the mannitol concentration of the medium altered the PD within a few minutes, and new stable values of PD were reached within 20 minutes after the osmotic change. With mannitol as the osmoticum, the most negative PD was measured at an external osmotic potential of -0.70 megapascals (MPa) for S₁ and S₂, while the most negative was at -0.40 MPa for S₃. Ethylene glycol, a permeant osmoticum, had little effect on PD, showing that the PD was sensitive to turgor, not to solute potential per se. For S₁ and S₂, the PD was less negative for turgor potentials lower than 0.1 MPa or greater than 0.3 MPa. S₃ samples exhibited a different turgor dependence, with a sharp optimum of the negativity of the PD at 0.3 MPa. The data are consistent with the proposal that the proton pump acts as a transducer of the osmotic conditions. They show that the osmotic sensitivity of the PD of mesocarp cells of broadbean changes with the stage of development of the pod.     

Effect of surface potential on Rb+ uptake in yeast: The effect of pH

The apparent K_m of Rb⁺ uptake and the zeta potential of yeast cells are appreciably affected by changes in the pH, variation of the concentration of the buffer cation Tris⁺ and addition of Ca²⁺ to the suspending medium. Irrespective of the way in which the zeta potential is affected, a direct relationship between the apparent K_m of the Rb⁺ uptake and the zeta potential is observed. A reduction of 8 mV in the zeta potential is accompanied by a 20-fold increase in the apparent K_m, which illustrates that electrostatic effects in ion uptake cannot be ignored. Measured zeta potentials are, to a good approximation, linearly related to surface potentials evaluated from a kinetic analysis of the Rb⁺ uptake. This shows the practical use of the zeta potential as a measure of the surface potential in studies of electrostatic effects in ion uptake by yeast. It is concluded that Tris⁺ and the aikaline earth cations inhibit the Rb⁺ uptake in yeast exclusively via a reduction in the surface potential. Protons, in addition, exert a competitive inhibition.     

Effects of perfluorooctane sulfonate on action potentials and currents in cultured rat cerebellar Purkinje cells

Recently, PFOS was reported to be ubiquitously detected in the environment, as well as in human serum, raising concerns regarding its health risks. We investigated the effects of PFOS on action potentials and currents in cultured rat cerebellar Purkinje cells using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the action potential frequency during current injection, the maximum rate of fall and the threshold of action potential, and negatively shifted the resting membrane potential at doses over 30microM. In voltage-clamp experiments, PFOS shifted the half-activation and inactivation voltages of I(Ca), I(Na), and I(K) toward hyperpolarization at 30microM. I(HCN1) expressed in Xenopus oocytes was similarly affected. Incorporation of PFOS into the cell membrane probably increased the surface negative charge density, thereby reducing the transmembrane potential gradient and resulting in hyperpolarizing shifts of both the activation and inactivation of ionic channels. These findings indicate that PFOS may exhibit neurotoxicity.     

Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress

The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media. With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV. With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability. The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy. Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth. L. acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol.     

Effects of the surface wettability and zeta potential of bioceramics on the adhesiveness of anchorage-dependent animal cells

Using calcium phosphate ceramics that have high biocompatibility with the living body, the effects of the surface characteristics of the bioceramics on cell adhesiveness were investigated. In the case of carriers with contact angles from 35° to 60°, the cell adhesiveness increased according to the increase in the wettability. Measurement of the zeta potentials of HAP-TCP sinters showed that these bioceramics had negative potentials from −2 mV to −6 mV. Electrochemical analysis suggested that the initial cell anchoring ratio (R_ia) and adhesive strength (F_a,enz) were affected by the surface ionic condition of the ceramic material. To clarify the effects of the surface potential of the ceramics on cell adhesiveness, the ceramic surface was modified chemically by means of various silane coupling reagents. The surface potential was regulated from −20 mV to +24 mV. Using these ceramics, the affinity and adhesiveness of the cells to the ceramics were found to be dominantly regulated by the surface potential. A negative potential was effective in increasing the adhesiveness, even though living cells have negative charges.     

Resting potential of the mouse neuroblastoma cells: II. Significant contribution of the surface potential to the resting potential of the cells under physiological conditions

In the previous paper, we showed that the K⁺ channels of the mouse neuroblastoma cell (clone N-18) are closed at low concentration of external K⁺ ([K⁺]₀) including the physiological concentration for the cells. In the present study, the origin of the resting membrane potential of N-18 cells has been examined. (1) The resting membrane potential of N-18 cells was depolarized by increasing concentration of the polyvalent cations (La³⁺, Fe³⁺, Co²⁺, Ca²⁺, Sr²⁺, Mg²⁺) and by decreasing the pH of the medium. The input membrane resistance was slightly increased during the depolarization. The depolarization was not explained in terms of the diffusion of the cations across the membrane, since the trivalent cations of greater ionic size were effective at much lower concentrations than the divalent cations. The results obtained from the measurements of ⁸⁶Rb efflux suggested that the depolarization cannot be explained in terms of blocking of the K⁺ channels by the cations. (2) An increase in Ca²⁺ concentration from 0.3 to 1.8 mM induced depolarization of about 10 mV at low [K⁺]₀ where the K⁺ channels are closed, but did not induce any depolarization at high [K⁺]₀ where the channels are open. (3) In order to estimate the changes in the zeta-potential, the electrophoretic mobility of N-18 cells was measured under various conditions. There was a close correlation between the changes in the zeta-potential and those in the membrane potential in response to the polyvalent cations and proton. On the other hand, an increase in K⁺-concentration in the medium, which induced a large depolarization in the cells, did not affect the zeta-potential. (4) The results obtained were explained by an electrical circuit model for the membranes of N-18 cells. In this model, an electrical circuit for the membrane part carrying no selective ionic channels, in which changes in the surface potential directly affect the transmembrane potential, is connected in parallel to the usual circuit model representing selective ionic channel systems. It was concluded that the surface potential contributes significantly to the resting membrane potential of N-18 cells at low [K⁺]₀ where the K⁺ channels are closed.     

Surface potential on the periplasmic side of the photosynthetic membrane of Rhodopseudomonas sphaeroides

Membrane surface potential on the periplasmic side of the photosynthetic membrane was estimated in cells, spheroplasts and chromatophores of Rhodopseudomonas sphaeroides. When the membrane potential (potential difference between bulk aqueous phases) was kept constant in the presence of carbonylcyanide m-chlorophenylhydrazone, addition of salt to a suspension of cells or spheroplasts induced a red shift in the carotenoid absorption spectrum which indicated a change in the intramembrane electrical field. The spectral shift is explained by a rise in electrical potential at the outside surface of the photosynthetic membrane due to a decrease in extent of the negative surface potential.The spectral shift occurred in the direction opposite to that in chromatophores, indicating that the sidedness of the membrane of cells or spheroplasts is opposite to that of chromatophores. The dependences of the extent of the potential change on concentration and valence of cations of salts agreed with the Gouy-Chapman relationship on the electrical diffuse double layer. The charge density on the periplasmic surface of the photosynthetic membrane was estimated to be ?2.9 · 10^?3 elementary charge per Ų, while that on the cytoplasmic side surface was calculated as ?1.9 · 10^?3 elementary charge per Ų (Matsuura, K., Masamoto, K., Itoh, S. and Nishimura, M. (1979) Biochim. Biophys. Acta 547, 91–102). Surface potential on the periplasmic side of the photosynthetic membrane was estimated to be about ?50 mV at pH 7.8 in the presence of 0.1 M monovalent salt.     

Membrane transport of polysialic acid chains: modulation of transmembrane potential

Polysialic acid (polySia) is a long polyanionic polymer (with the degree of polymerization, DP, up to 200) of negatively charged sialic acid monomers. PolySia chains are bound to the external surface of some neuroinvasive bacterial cells and neural cells. PolySia serves as a potent regulator of cell interactions via its unusual biophysical properties. In the present paper, the analysis, based on the Goldman-Hodgkin-Katz equation, of transmembrane potential changes resulting from transmembrane translocation of polySia is performed. The relationships between the transmembrane potential and the polySia DP (up to 200), the temperature, the cation/ anion permeability ratio, and the inner/outer concentration ratio of polySia has been plotted and discussed. The maximal membrane potential changes, up to 118 mV, were found for a permeability ratio greater than one. The increase of the polySia chain length resulted in the diminution of this effect. The temperature-dependent changes in membrane potential were less than 7 mV in the range 0-50 degrees C. The change in the concentration ratio (into its reciprocal) resulted in a mirror reflection of the membrane potential curves. The results show that the expression of polySia chains in bacterial cells can be responsible for the modulation of the transmembrane potential of the bacterial inner membrane. We suggest that the polySia chains can influence the transmembrane potential of neural cell membranes in a similar way. This analysis also describes the effect of the transmembrane translocation of negatively charged polyanionic polynucleotydes on the cell membrane potential.     

Intrinsic electric fields and membrane bending

The fragmentation of human erythrocytes heated in a range of ionic environments has been examined by video microscopy, , the average number of surface wave crests growing on the cell rim during fragmentation by membrane externalization, andI, the percentage of cells internalizing membrane, were scored.The membrane diffusion potential was altered experimentally on decreasing the extracellular chloride concentration by substituting either membrane-impermeant sorbitol or Na gluconate for some NaCl. The external-membrane-face surface potential was altered either by surface charge depletion or by ionic strength changes. The dependence of morphological change on diffusion potential at constant cell volume and surface potentials was established over a 34-mV change in diffusion potential. The rate constants for morphological change with charge depletion at different diffusion potentials are largely independent of the diffusion potential. A l.O-mV increase in diffusion potential has an effect on morphological change of comparable magnitude to that of a 1.0-mV decrease in the modulus of the negative surface potential. When the diffusion potential increased on decreasing both the extracellular diffusible ion concentration and extracellular ionic strength, the effect on cell morphology of increasing the modulus of the surface potential was overcome by the effects of the diffusion potential change.     

Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells

During storage, red blood cells (RBCs) for transfusion purposes suffer progressive deterioration. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface which creates a repulsive electrical zeta potential. These charges help prevent the interaction between RBCs and other cells, and especially among each RBCs. Reports in the literature have stated that RBCs sialylated glycoproteins can be sensitive to enzymes released by leukocyte degranulation. Thus, the aim of this study was, by using an optical tweezers as a biomedical tool, to measure the zeta potential in standard RBCs units and in leukocyte reduced RBC units (collected in CPD-SAGM) during storage. Optical tweezers is a sensitive tool that uses light for measuring cell biophysical properties which are important for clinical and research purposes. This is the first study to analyze RBCs membrane charges during storage. In addition, we herein also measured the elasticity of RBCs also collected in CPD-SAGM. In conclusion, the zeta potential decreased 42% and cells were 134% less deformable at the end of storage. The zeta potential from leukodepleted units had a similar profile when compared to units stored without leukoreduction, indicating that leukocyte lyses were not responsible for the zeta potential decay. Flow cytometry measurements of reactive oxygen species suggested that this decay is due to membrane oxidative damages. These results show that measurements of zeta potentials provide new insights about RBCs storage lesion for transfusion purposes.     

Measurement of surface potential and surface charge densities of sarcoplasmic reticulum membranes

Summary The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS^–) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS^– is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(e₀/kT, where ₀ is the membrane surface potential,e is the electronic charge, andkT has its usual meaning. The potential measured is characteristic of the ANS^– bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS^–, referred to as the aqueous phase bathing the surface, were in the range –10 to –15 mV. This was observed for the outside and inside surfaces of the Ca²⁺-ATPase-rich fraction of theSR and for both surfaces of theSR fraction rich in acidic Ca²⁺ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS^– binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca²⁺ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca²⁺ permeability.The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types ofSR. Values for the Ca²⁺-ATPase richSR fraction were between 2.9×10³ and 3.8×10³ esu/cm², (0.96×10^–6 and 1.26×10^–6 C/cm²) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6×10³ and ca. 2.2×10³ esu/cm² (2.2×10^–6 and 0.73×10^–6 C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in theSR. The acidic Ca²⁺ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of Å from the membrane surface. The experiments gave evidence for a Donnan effect on the K⁺ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in thisSR fraction.The equilibrium constant for ANS^– was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in theSR lumen.     

Membrane potential changes associated with pinocytosis of serum lipoproteins in L cells

L cells exhibit spontaneous oscillations of membrane potential in accord with fluctuations of the cytoplasmic Ca²⁺ concentration. Upon addition of low-density lipoprotein (LDL), L cells show a prolonged hyperpolarization which is followed by an increase in the frequency of membrane potential oscillations. These membrane potential changes induced by LDL were inhibited by Ca²⁺-channel blockers. LDL-induced membrane potential changes were accompanied by a vigorous pinocytosis which was coupled with the formation of ring-like ridge structures on the cell surface. These electrical and morphological changes were also induced by high-density lipoprotein (HDL) but not by very-low-density lipoprotein (VLDL). These results suggest that the application of LDL or HDL to the membrane surface elicits a rapid influx of Ca²⁺ into the cytosol, resulting in membrane hyperpolarization. A rise in cytoplasmic Ca²⁺ may be implicated in the primary factor for the pinocytic process.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.