Article

# Divalent Ions and the Surface Potential of Charged Phospholipid Membranes

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## Abstract

Phospholipid bilayer membranes were bathed in a decimolar solution of monovalent ions, and the conductance produced by neutral carriers of these monovalent cations and anions was used to assess the electric potential at the surface of the membrane. When the bilayers were formed from a neutral lipid, phosphatidylethanolamine, the addition of alkaline earth cations produced no detectable surface potential, indicating that little or no binding occurs to the polar head group with these ions. When the bilayers were formed from a negatively charged lipid, phosphatidylserine, the addition of Sr and Ba decreased the magnitude of the surface potential as predicted by the theory of the diffuse double layer. In particular, the potential decreased 27 mv for a 10-fold increase in concentration in the millimolar-decimolar range. A 10-fold increase in the Ca or Mg concentration also produced a 27 mv decrease in potential in this region, which was again due to screening, but it was necessary to invoke some specific binding to account for the observation that these cations were effective at a lower concentration than Ba or Sr. It is suggested that the ability of the alkaline earth cations to shift the conductance-voltage curves of a nerve along the voltage axis by 20–26 mv for a 10-fold increase in concentration may be due to essentially a screening rather than a binding phenomenon.

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... Because choline is charged, choline addition also increases saline ionic strength. Increases in ionic strength can increase the depolarization required to achieve a given level of channel opening (Brismar 1973;Hille 1992;Hille et al. 1975;McLaughlin et al. 1971;Miedema 2002). This effect would result in our depolarizations to 0 mV inducing less voltageactivated potassium and calcium channel opening, and thus decreased outward currents. ...
... An alternative explanation of choline's actions is that it activates ACh receptors that reduce outward current. Choline activates and/or inhibits nicotinic and muscarinic cholinergic receptors in multiple systems (Alkondon et al. 1997; Barry and Gelperin 1982;Dale 1914;Gardner et al. 1984;Kilbinger and Kruel 1981;Krnjević and Reinhardt 1979;Lape et al. 2009;McLaughlin et al. 1971). Pyloric neurons and Retzius cells Fig. 7. Adding sucrose to increase normal saline osmolarities to those present in the high-choline hydroxide salines either increased or had no effect on outward currents. ...
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... These values are equivalent to 1 e/nm 2 -thus equal or beyond those observed biologically or used experimentally. At the surface of biological membranes, the σ w has been estimated at 40-160 mC/m 2 or 0.25 to 1 e/nm 2 [51,52], and at the surface layer of SiO 2 membrane at 26 mC/m 2 [53]. As expected the σ w influences the ion concentrations greatly near the charged pore wall or disk surfaces. ...
Article
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... As noted in prior publications (Smith and Trimarchi, 2001;Molina et al., 2004;Kreitzer et al., 2007Kreitzer et al., , 2017, the signals generated by the self-referencing H + -selective microelectrodes used in these experimental conditions are not likely to arise from surface potentials or other stray sources of extracellular voltages. For example, extracellular voltage fields generated by isolated cells are usually in the nanovolt range and below the sensitivity of ion-selective self-referencing probes (Kuhtreiber and Jaffe, 1990;Smith et al., 1999), and electrical potentials from local boundary conditions associated with membrane surface charges (McLaughlin et al., 1971(McLaughlin et al., , 1981 drop with the Debye length and do not extend into the medium by more than tens of angstroms (cf. Cevc, 1990); our H + -selective microelectrodes were located at least 1 µm away from the surface of cells. ...
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... Lampert [17] has discussed use of nonlinear P-B equation. It may be added here that McLaughlin et al. [18] experimentally showed that predictions of linearized PB theory agreed adequately with GCS (Gouy-Chapman-Stern) theory for incorporating adsorption in the electrical double layer. Nonlinear P-B becomes important when charged dielectric planar wall-colloid potentials needed to be considered. ...
Article
Stability of hydrophobic colloids is an important phenomenon in colloid science. The hydrophobic colloids are known to get destabilized by the counterions of electrolytes leading to coagulation guided by Schulze–Hardy (S-H) rule. There are exceptions to this rule; effective improvement has been attempted but not with good success. Recently, the role of coions in the coagulation process was considered: an inverse Schulze–Hardy (iS-H) rule has been proposed. We have discussed the basics of colloid solutions, and colloid stability: also have tested the S-H and iS-H rules in terms of a number of positively and negatively charged hydrophobic colloids. Critical coagulation concentrations, zeta-potential, charges of coions and counterions, and ionic strengths have been separately (as well as together) considered to correlate the coagulation phenomenon. Partial agreements with the rules are only found. The article summarizes the fundamentals of the field of colloid stability, and its current status. Analysis of experimental findings in a new perspective (in terms of a new analytical procedure) has been proposed with a view to its projection toward future studies.
... These very low affinities are similar to the binding affinities between phospholipids and various cations documented in the literature [39,41]. We thus suspected that divalent cations may bind to membrane phospholipids and mediate the effects by altering the membrane surface potential [43,44,56]. To examine whether the potentiation of TMEM16A current by divalent cations involves phospholipids, we conducted several lines of experiments. ...
Article
Full-text available
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... Previous experimental research suggested the use of well-characterized liposome suspensions and electrokinetic measurements to control the charge of these colloid particles [9,10]. The relatively simple Gouy-Chapman-Stern (GCS) model describes the electric potential distribution at the membrane-water interface considering monovalent ion adsorption [11], and was verified by several methods [12]. This model assumes that the electric potential is distributed uniformly in a lateral direction. ...
Article
The adsorption of large polycations on a charged lipid membrane is qualitatively different from the small inorganic cations, which almost uniformly populate the membrane surface. We assume that the polycationic adsorption layer might be laterally inhomogeneous starting from a certain polymer length, and this effect can be more visible for membranes with low anionic lipid content. To study systems with inhomogeneous adsorption layers, we carried out electrokinetic measurements of mobility of liposomes containing anionic and neutral phospholipids in the presence of polylysine molecules. Some of these systems were simulated by all-atom molecular dynamics. Here we proposed a theoretical approach accounting for the formation of separated regions at the membrane surface, which differ in charge density and surface potential. Our model allowed us to determine the adsorption layer’s geometric parameters such as surface coverage and surface-bound monomer fraction of polymer, which correlate with the molecular dynamics (MD) simulations. We demonstrated that the configurations polylysine adopts on the membrane surface (tall or planar) depends on the polymer/membrane charge ratio. Both theory and MD indicate a decrease in the anionic lipid content, alongside with a decrease in the bound monomer fraction and corresponding increase in the extension length of the adsorbed polymers.
... The increase in excitatory synaptic outputs resulting from the increased firing activity may produce glutamate spillover from excitatory synapses, and this may further enhance the excitability of adjacent neurons through its actions on extrasynaptic glutamate receptors including both ionotropic and metabotropic ones [59]. The fall in extracellular Ca 2+ concentration is due to massive Ca 2+ entry into neurons during excessive neuronal firing and synaptic activities, and this may depolarize adjacent neurons through reducing the surface potential of the plasma membrane [52]. The relative contributions of these changes in extracellular K + , excitatory synaptic outputs, glutamate, and Ca 2+ to the induction of ictal discharges remain elusive, because the changes interact with each other [66]. ...
Article
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The intracellular Cl− concentration ([Cl−]i) is tightly regulated in brain neurons for stabilizing brain performance. The [Cl−]i in mature neurons is determined by the balance between the rate of Cl− extrusion mainly mediated by the neuron-specific type 2 K+-Cl− cotransporter (KCC2) and the rate of Cl− entry through various Cl− channels including GABAA receptors during neuronal activity. Disturbance of the balance causes instability of brain circuit performance and may lead to epileptic seizures. In the first part of this review, we discuss how genetic alterations in KCC2 in humans cause infantile migrating focal seizures, based on our previous report and others. Depolarization of the membrane potential increases the driving force for Cl− entry into neurons. Thus, the duration of action potential spike generation and the frequency of excitatory synaptic inputs are the crucial factors for determining the total amount of Cl− entry and the equilibrium [Cl−]i in neurons. Moreover, there is also a significant interdependence between the neuronal activity and the KCC2 expression. In the second part, we discuss plausible mechanisms by which excessive neuronal activity due to excitotoxic brain insults or other epilepsy-associated gene mutations may cause the Cl− imbalance in neurons and lead to epileptic discharges over the brain, using the schematic “unifying foci” model based on literature.
... Lampert [17] has discussed use of nonlinear P-B equation. It may be added here that McLaughlin et al. [18] experimentally showed that predictions of linearized PB theory agreed adequately with GCS (Gouy-Chapman-Stern) theory for incorporating adsorption in the electrical double layer. Nonlinear P-B becomes important when charged dielectric planar wall-colloid potentials needed to be considered. ...
Article
The Schulze–Hardy (S-H) rule that explains the precipitation (flocculation/coagulation) of colloid particles is almost 125 years old. This rule states that counter ions of the electrolytes are responsible for the precipitation of the charged colloids, and the charge on the counter ion determines the amount of the electrolyte required for the event. Though this is necessarily true, it does not take into account various other variables in the system viz. charges on the co-ions, the ionic strength of the solution, the charge density on the interacting ions, and that of the colloid particles, shape of the particles, etc. In this article, we have modified the S-H rule taking into account some of the above mentioned variables viz. the ionic strength of the solution, the charge density of the interacting ions as well as that of the associated colloid particles. We have attempted to find out the validity of the modified S-H rule on the nano, micro, and macro colloidal systems so far remaining unreported in the literature.
... This is similar or goes beyond values measured biologically or tested experimentally. Surface charge density of biological membranes ranges from 40 to 160 mC/m 2 (McLaughlin et al. 1971). In the experimental study the σ o of carboxylated polystyrene particles was 64 mC/m 2 (Qiu et al. 2015). ...
... 8,9 Because of the importance of ion-membrane interactions, they have been studied extensively using fluorescent methods. [10][11][12][13][14][15] While these methods can generate important insights, the fluorophore itself, or the way in which the measurement is conducted/interpreted, might obscure or influence the molecular level mechanisms that are at play. For example, it was shown only recently that divalent cations such as Ca 2+ , Mg 2+ , and Ba 2+ do not homogeneously bind to freely suspended charged lipid membranes, but instead form transient domains of ion-lipid complexes. ...
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The interaction of divalent copper ions (Cu²⁺) with cell membranes is crucial for a variety of physiological processes of cells such as hormone synthesis and cellular energy production. These interactions would not be possible without membrane hydration. However, the role of water has not received a lot of attention in membrane studies. Here, we use high-throughput wide-field second harmonic (SH) microscopy to study the interaction between Cu²⁺ and hydrated free standing Montal-Müller lipid membranes. The symmetric lipid membranes are composed of 1,2-diphytanoyl-sn-glaycero-3-phosphocholine (DPhPC) and either 1,2-diphytanoyl-sn-glycero-3-phosphate (DPhPA) or 1,2-diphytanoyl-sn-glycero-3-phospho L-serine (DPhPS), and are brought in contact with divalent Cu²⁺, which are added to one leaflet, while maintaining the ionic strength balance. We observe transient domains of high SH intensity. In these areas Cu²⁺ ions bind to the charged head groups leading to charge neutralization on one side of the membrane. This exposes the ordered water at the non-interacting side of the membrane interface, which can be used to compute the interfacial membrane potential difference. We find that domains of lipids with PA head groups display a higher interfacial membrane potential than those with PS headgroups, which converts into higher dynamic electrostatic free energies and binding constants.
... The increased K + concentration presumably depolarizes the resting membrane potential via a less negative K + Nernst potential; the resting neuronal membrane is primarily permeable to K + . The reduced concentration of Mg 2+ and Ca 2+ may enhance excitability through reduced charge screening of the neuronal membrane, resulting in a negative shift in the activation curves for voltagedependent conductances (Frankenhaeuser and Hodgkin, 1957;McLaughlin et al., 1971). These ionic concentrations afford a critical level of excitability in the in vitro recurrent cortical network such that reverberant activity can be sustained in rhythmic cycles. ...
Article
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During even the most quiescent behavioral periods, the cortex and thalamus express rich spontaneous activity in the form of slow (<1 Hz), synchronous network state transitions. Throughout this so-called slow oscillation, cortical and thalamic neurons fluctuate between periods of intense synaptic activity (Up states) and almost complete silence (Down states). The two decades since the original characterization of the slow oscillation in the cortex and thalamus have seen considerable advances in deciphering the cellular and network mechanisms associated with this pervasive phenomenon. There are, nevertheless, many questions regarding the slow oscillation that await more thorough illumination, particularly the mechanisms by which Up states initiate and terminate, the functional role of the rhythmic activity cycles in unconscious or minimally conscious states, and the precise relation between Up states and the activated states associated with waking behavior. Given the substantial advances in multineuronal recording and imaging methods in both in vivo and in vitro preparations, the time is ripe to take stock of our current understanding of the slow oscillation and pave the way for future investigations of its mechanisms and functions. My aim in this Review is to provide a comprehensive account of the mechanisms and functions of the slow oscillation, and to suggest avenues for further exploration.
... This is similar or goes beyond values measured biologically or tested experimentally. Surface charge density of biological membranes ranges from 40 to 160 mC/m 2 (McLaughlin et al. 1971). In the experimental study the σ o of carboxylated polystyrene particles was 64 mC/m 2 (Qiu et al. 2015). ...
Article
Full-text available
Understanding the physics of object translocation in nanopores is critical for using nanopores as sensors of molecular properties and as object size and shape sensors. Based on Poisson-Nernst-Planck and Navier–Stokes simulations we dissect three axial pressures and forces at disk edges (upper, lower and rim) – Coulomb, dielectric and fluidic. Axial Coulomb and dielectric rim forces are small and cancel each other. Upper and lower axial forces are largely controlled by the external axial electric field and interestingly by the pore wall charges that determine the amplitude and direction of axial combined force. Axial total Coulomb force (sum of its upper and lower edge components) makes the greatest contribution, but the axial total dielectric force (calculated using Maxwell stress tensor), which opposes it is surprisingly large. External ion concentration alters Coulomb and axial dielectric forces but influences only their amplitude. Axial total fluidic force is near zero (its upper and lower disk edge components are significant but cancel each other) regardless of external electric field, but pore wall charges and external fluidic pressure can alter it. Modest changes of external electric field or concentration produce axial forces comparable to those produced by large external fluidic pressures. Axial forces depend little on disk’s axial position. Finally, mean axial pressures (calculated to compare forces acting on disks of different radius) are greater for larger disks.
Chapter
The surface charge of biological membranes is produced by dissociable groups of membrane constituents, mainly phospholipids and proteins. The surface potential (Ψo) is related to the surface charge density (6) according to the Gouy-Chapman equation which, when simplified for a symmetric electrolyte, is$${{\varphi }_{o}}=\frac{2RT}{zF}\arcsin h\left[ 6{{\left( 8RT{{\varphi }_{o}}{{\varphi }_{r}}C \right)}^{-\frac{1}{2}}} \right]$$ (1) where R is the gas constant, T is the absolute temperature, F is the Faraday constant, C denotes the concentration of the electrolyte, and z is its valency,∈o is the permittivity of the vacuum, and ∈r is the relative permittivity (dielectric constant) of the medium. Because of electric attraction or repulsion, the concentration of ions in the immediate vicinity of the membrane surface (Co) is different from that in the bulk solution (C∞), as described by the Boltzmann distribution$${{C}_{o}}={{C}_{\infty }}\exp \left( -zF{{\varphi }_{o}}/RT \right)$$ (2)
Chapter
Calcium influx through voltage-dependent Ca channels leads to a transient increase in intracellular Ca, which can initiate a number of biological processes, such as muscle contraction, synaptic transmission, secretion, ciliary motility, enzyme activation, growth and development. The electrical activity promoted by Ca channels is the most variable and widespread form of excitation. Ca channels are ubiquitous, from unicellular organisms to mammals, and Ca-dependent electrogenesis results in membrane depolarizations of wide range in amplitude and duration. Currents through Ca channels are diverse in their properties, and they can have great impact on the Ca homeostasis in cells. During the influx of Ca ions through open channels the initially low intracellular Ca activity of about 0.1 uM may increase several orders of magnitude within milliseconds.
Chapter
A photopigment extracted from the honeybee compound eye was incorporated into positively charged lipid bilayers, in the dark. As a consequence cation selective pathways are formed, as deduced from conductance and potential measurements. Light causes a further increase of macroscopic conductance, associated with the formation of ionic channels with individual conductance of about 80.
Article
Fluorescent permeant charged probes are commonly used for monitoring the trans-membrane potential in lipid vesicles and biological membranes, which has been earlier described by various mathematical models. In the present study, we developed a more complex model based on the computational step-by-step analysis of the influence of various factors, such as the membrane surface potential, ionic strength, and the aggregation properties of cationic cyanine probe DiSC3(5) in the membrane and aqueous phases, in addition to the Nernstian distribution of the probe across the membrane and the hydrophobic interaction with the lipid bilayer. The final full model allows prediction of the optimal experimental conditions for monitoring the trans-membrane potential, such as the probe/lipid ratio and the concentration of liposomes, with a given percentage of negatively charged phospholipids in the membrane, the ionic strength of the aqueous media, the "membrane-water" partition coefficient and the aggregation properties of the probe, as well as the most adequate mode of fluorescence measurement. In agreement with many experimental studies, this model showed high voltage sensitivity of the quantity of the aqueous phase DiSC3(5) monomers, showing its almost exponential decrease with an increase in the trans-membrane potential value. The model also demonstrated the highest voltage sensitivity of the ratio of the quantity of DiSC3(5) monomers in the aqueous phases to that in the membrane phase. A new combined parameter, the logarithmic function of this ratio, demonstrated almost linear changes within a wide range of the trans-membrane potential changes.
Chapter
Cardiac contraction is produced by a transient increase in intracellular calcium concentration ([Ca2+]i) which is initiated by surface membrane depolarization during the action potential (for review see Bers, 1991). Although many processes are involved in the link from excitation to contraction, excitation-contraction coupling (E-C coupling) is generally considered to refer to the processes that lead to an increase in [Ca2+]i after depolarization since the contractile activation is secondary to the increase in [Ca2+]i acting via troponin on thin (actin) filaments (e.g. Holroyde et al., 1980 for reviews see Bers, 1991; Ruegg, 1992). New & Trautwein (1972) proposed that during normal cardiac E-C coupling calcium influx across the surface membrane triggers a larger calcium release from intracellular stores (the sarcoplasmic reticulum-SR). This occurs via the ‘calcium-induced calcium release’ (CICR) mechanism (Fabiato, 1983;1985) which remains the cornerstone of E-C coupling to this day.
Chapter
The arrangement of lipids, proteins and water in a membrane is intrinsically dynamic, changing to suit its instantaneous functional needs: therefore some insight on the control mechanisms of membrane functions can be obtained from the comprehension of the dynamic properties of its components.
Chapter
Changes in P-31 chemical shifts and relaxation times demonstrate that Ca2+ binds to the phosphate group of phosphatidylserine (PS) and restricts the motion of this group more than does Mg2+. Smaller differences in P-31 shifts and relaxation occur if tetramethylammonium is substituted for Na+ or the bulk concentration of Na+ is decreased. These latter changes support the conclusion from a Na-23 relaxation rate study that Na+ binds specifically to the PS headgroup.
Article
Excellent progress has been made toward understanding the physiology and pharmacology of specific calcium-related cellular processes of the brain, but few studies have provided an integrated view of brain calcium kinetics. To further the knowledge of the size and binding properties of brain calcium compartments, the authors have conducted a series of experiments in hippocampal brain slices exposed to high and low extracellular calcium. Slices were incubated in buffers containing 0.001 to 4.5 mmol/L calcium for up to 75 minutes. Slice calcium content was analyzed by three methods: exchange equilibrium with Ca-45, synchrotron-radiation-induced x-ray emission, and inductively coupled plasma. Data were analyzed using a model based on a Langmuir isotherm for two independent sites, with additional extracellular and bound compartments. In parallel experiments, altered low calcium had no effect on slice histology and only mild effects on slice adenylates. When combined with prior Ca-45 and fluorescent probe binding experiments, these results suggest that there are at least five kinetically distinct calcium compartments: (1) free extracellular (similar to10%); (2) loosely associated extracellular plasma membrane (similar to55%); (3) intracellular compartment with moderate avidity (similar to17%); (4) tightly bound, nonexchangeable intracellular compartment (similar to15%); and (5) free cytoplasmic (<0.01%). If only the third compartment is considered a potential calcium buffer, then the buffering ratio is calculated to be similar to2,700:1, but if the second compartment is also included, then the buffering ratio would be similar to13,000:1. This may explain the wide range of estimates observed by fluorescent probe studies.
Chapter
During neuronal hyperactivity, including repetitive stimulations, applications of excitatory amino acids, epilepsy, spreading depressions and anoxia, large changes in the concentration of extracellular ion occur. Such changes are accompanied by changes in the extracellular space (ES) size, which can shrink by up to 60%. The changes in ES size can be rapid, localized and also rapidly reversible. Several mechanisms can account for the ES changes: KC1 uptake in glia, the glial spatial buffering, NaCl uptake into neurons, metabolic increase of the intracellular osmolarity. The observed ES changes may have a number a consequences, among which the maintenance of neuronal functioning appears to be the most important.
Chapter
Leakage induced across the plasma membrane of cells by poreforming agents is prevented by divalent cations. The same is true of leakage from liposomes or across a planar lipid bilayer. Divalent cation sensitivity appears not to depend on binding either to agent or to lipid. This paradoxical result may reflect the occurrence of a phenomenon described as ‘surface conductance’
Article
Traditionally, it is considered that neuronal synchronization in epilepsy is caused by a chain reaction of synaptic excitation. However, it has been shown that synchronous epileptiform activity may also arise without synaptic transmission. In order to investigate the respective roles of synaptic interactions and nonsynaptic mechanisms in seizure transitions, we developed a computational model of hippocampal cells, involving the extracellular space, realistic dynamics of [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text] ions, glial uptake and extracellular diffusion mechanisms. We show that the network behavior with fixed ionic concentrations may be quite different from the neurons' behavior when more detailed modeling of ionic dynamics is included. In particular, we show that in the extended model strong discharge of inhibitory interneurons may result in long lasting accumulation of extracellular [Formula: see text], which sustains the depolarization of the principal cells and causes their pathological discharges. This effect is not present in a reduced, purely synaptic network. These results point to the importance of nonsynaptic mechanisms in the transition to seizure.
Article
Herein, we demonstrate that Cu(2+) binds bivalently to phosphatidylethanolamine (PE), the second most abundant lipid in mammalian cells. The apparent equilibrium dissociation constant, KDApp, for the Cu(2+)-PE complex at physiological pH is approximately 2 μM and is insensitive to the concentration of PE in the membrane. By contrast, at pH 10.0, where PE lipids bear a negative charge, KDApp decreases with increasing PE content and has a value of 150 nM for bilayers containing 70 mol % PE. The oxidation of double bonds in PE-containing bilayers can be monitored in the presence of Cu(2+). Strikingly, it was found that the oxidation rate is 8.2 times faster at pH 7.4 for bilayers containing 70 mol % PE than for pure phosphatidylcholine (PC) bilayers upon exposure of both to 70 μM Cu(2+) and 10 mM hydrogen peroxide. The rate of oxidation increases linearly with the PE content in the membrane. These results may help explain the high level of lipid oxidation in PE-containing membranes for neurodegenerative diseases and autism where the Cu(2+) concentration in the body is abnormally high.
Chapter
An unambiguous understanding of ion permeation, selectivity, and their relation to molecular structure and function requires detailed information about the energy profiles encountered by ions during transport [15,25]. Here, we use Eyring rate theory approximations [19] to develop simplified models for the energy profiles encountered by monovalent ions crossing gramicidin A [17,28] or acetylcholine receptor (AChR) channels [5, 34]. The energy profiles are assessed by fitting simple barrier models to open-channel current — voltage (I-V) relations obtained over a wide range of symmetrical, single electrolyte concentrations. First we describe the I-V behavior for Li, Na, K, Rb, and Cs in the gramicidin channel using a three-barrier, four-site (3B4S) model [58]. The particular 3B4S″ variant [58] of this model describes the electrical data well [16] and is consistent with the accepted tertiary structure proposed [61] from the known primary sequence of gramicidin [59]. Then, initial I-V relations for Na [11] and Cs (J. A. Dani and G. Eisenman, in preparation) in the AChR channel are described in terms of two-barrier, one-site (2B1S) models. Some indadequacies of the classical 2B1S model [1, 30, 44, 45] are removed by allowing the outer barrier or the site to have width. The studies presented here represent a start in our exploration of the range of possible energy profiles than can account for the data. We hope that they will give information that can ultimately be matched with structural models [13, 22, 38, 53] based on the primary sequences of the AChR’s subunits [6, 9,13, 51–53]. Eventually, as both permeation and structure are more clearly understood, they can be integrated into the overall function of the AChR.
Chapter
Although it has been widely hypothesized that in the central nervous system of mammals the chemical composition of the extracellular fluid is maintained constant through precise regulatory mechanisms, it has become increasingly clear in the last 20 years that significant changes may occur during intense neuronal activity. In this respect, the development of ion-selective microelectrodes (Ammann, 1986) has been a decisive factor to determine the precise nature and extent of the possible ionic changes in the extracellular space. Indeed, it has long been known that nerve cell function is essentially underlain by transmembrane ionic currents, inwardly or outwardly directed, induced either through changes in membrane potential or the action of neurotransmitters. Therefore, any sustained activity of nerve cell could, in principle, alter the ion content of the extracellular or intracellular compartments. Thus, it has been shown that intense neuronal activity is associated with changes in the concentration of extracellular potassium ([K+]o), sodium ([Na+]o), calcium ([Ca2+]o), magnesium ([Mg2+]o), and chloride ([Cl−]o) ions and in pH (Benninger et al., 1980; ten Bruggencate et al., 1976; Dietzel et al., 1982; Futamachi et al., 1974; Heinemann and Lux, 1975; Kraig and Nicholson, 1978; Lux and Neher, 1973; Morris and Krnjevic, 1973; Nicholson et al., 1978; Prince et al., 1973; Pumain and Heinemann, 1985; Somjen, 1979, 1980; Sykova et al., 1976; Urbanics et al., 1978). The magnitude of the ion changes in the extracellular space depends not only on the transmembrane ionic fluxes but also on the volume of distribution of these ions and on how they migrate in the extracellular space.
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The emerging technological revolution in genetically encoded molecular sensors and super-resolution imaging provides neuroscientists with a pass to the real-time nano-world. On this small scale, however, classical principles of electrophysiology do not always apply. This is in large part because the nanoscopic heterogeneities in ionic concentrations and the local electric fields associated with individual ions and their movement can no longer be ignored. Here, we review basic principles of molecular electrodiffusion in the cellular environment of organized brain tissue. We argue that accurate interpretation of physiological observations on the nanoscale requires a better understanding of the underlying electrodiffusion phenomena.
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Chlorhexidine (CHX) is an antibacterial agent used in various types of pharmaceutical products. Therefore, CHX is easily found around us. Owing to its positive charge, the electrochemical property of cell membranes was assumed to be a key point of cytotoxic action of CHX. Depolarization of membranes attenuated the cytotoxic action of CHX in rat thymic lymphocytes. CHX interfered with annexin V binding to membranes. Manipulations to induce exposure of phosphatidylserine on the outer membrane surface augmented the cytotoxic action of CHX, indicating that changes in the electrochemical property of membranes affected the cytotoxic action of CHX. Hence, CHX might kill cells physiologically undergoing apoptosis, resulting instead in necrotic cell death. However, the threshold CHX concentration in this in vitro study was slightly higher than blood CHX concentrations observed clinically. Therefore, these results may support the safety of CHX use although CHX possesses unique cytotoxic actions described in this study.
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Ions interact with water via short-ranged ion-dipole interactions. Recently, an additional unexpected long-ranged interaction was found: The total electric field of ions influences water-water correlations over tens of hydration shells, leading to the Jones Ray effect, a 0.3% surface tension depression. Here, we report such long-range interactions contributing substantially to both molecular and macroscopic properties. Femtosecond elastic second harmonic scattering (fs-ESHS) shows that long-range electrostatic interactions are remarkably strong in aqueous polyelectrolyte solutions, leading to an increase in water-water correlations. This increase plays a role in the reduced viscosity, which changes more than two orders of magnitude with polyelectrolyte concentration. Using D 2 O instead of H 2 O shifts both the fs-ESHS and the viscosity curve by a factor of ~10 and reduces the maximum viscosity value by 20 to 300%, depending on the polyelectrolyte. These phenomena cannot be explained using a mean-field approximation of the solvent and point to nuclear quantum effects.
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Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F ⁻ containing internal solution together with a Ca ²⁺ containing external solution — so that a CaF 2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F ⁻ ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.
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The determination of the membrane surface charge is based on the measurement of the surface potential difference at both sides of the bilayer lipid membrane (BLM) connected with the asymmetrical concentration of the electrolyte in both solutions. In the short-circuit regime the intramembrane potential jump is caused by the difference in the two surface potentials. In order to find the intramembrane potential jump the BLM capacitance dependence on voltage was used. In some range of electrolyte concentrations a dependence of the potential jump on the surface charge was found. The charge density was calculated by applying the Gouy-Chapman theory of the diffuse double layer. Surface charges were determined for BLM of common bovine brain lipids, phosphatidylethanolamine, dioleyllecithin and azolectine.
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Ions are ubiquitous in nature. They play a key role for many biological processes on the molecular scale, from molecular interactions, to mechanical properties, to folding, to self‐organisation and assembly, to reaction equilibria, to signalling, to energy and material transport, to recognition etc. Going beyond monovalent ions to multivalent ions, the effects of the ions are frequently not only stronger (due to the obviously higher charge), but qualitatively different. A typical example is the process of binding of multivalent ions, such as Ca 2+ , to a macromolecule and the consequences of this ion binding such as compaction, collapse, potential charge inversion and precipitation of the macromolecule. Here we review these effects and phenomena induced by multivalent ions for biological (macro)molecules, from the “atomistic / molecular” local picture of (potentially specific) interactions to the more global picture of phase behaviour including, e.g., crystallisation, phase separation, oligomerisation etc. Rather than attempting an encyclopedic list of systems, we rather aim for an embracing discussion using typical case studies. We try to cover predominantly three main classes: proteins, nucleic acids, and amphiphilic molecules including interface effects. We do not cover in detail, but make some comparisons to, ion channels, colloidal systems, and synthetic polymers. While there are obvious differences in the behaviour of, and the relevance of multivalent ions for, the three main classes of systems, we also point out analogies. Our attempt of a comprehensive discussion is guided by the idea that there are not only important differences and specific phenomena with regard to the effects of multivalent ions on the main systems, but also important similarities. We hope to bridge physico‐chemical mechanisms, concepts of soft matter, and biological observations and connect the different communities further.
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The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-β-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.
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Lysozyme loaded niosomes containing various molar ratios of two kinds of surfactants were prepared and the properties of these niosomal formulations were studied. The results revealed that the size of niosomes varied between 240.06 ± 32.41 and 895.2 ± 20.84 nm. Formulations with the lowest size and no precipitation had entrapment efficiencies ranging from 60.644 ± 3.310 to 66.333 ± 1.98%. Their controlled release profiles after 48 h were 15.67, 20.67 and 31.50%. After 2 months, the most stable formulation in terms of size, PDI, zeta potential, and entrapment efficiency was used to study the secondary structures of lysozyme in niosomal and free forms. Lysozyme loaded niosome and lysozyme adsorbed on the surface of niosome fell into one category in terms of the formation of α-helix,β -sheet, and turn structures. This study suggests that niosomes could be a promising delivery system for lysozyme with prolonged release profiles, which can be used in pharmaceutical and food industries.
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In the past several decades there has been an increased awareness of the effects of “heavy” metals that act as pollutants of our environment. As greater technological advances in chemical detection and analysis of trace elements have been made, the more widespread has become the concern over exposure to these apparently ubiquitous metals (Chisolm, 1980). Since all elements are potentially toxic according to dose, almost all physiological systems are affected by pharmacological doses of trace elements, including the reticuloendothelial system.
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Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that is thought to participate in the regulation of many physiological processes and may play a key role in several diseases. Herein, we found that Cu²⁺ binds tightly to supported lipid bilayers (SLBs) containing S1P. Specifically, we demonstrated via fluorescence assays that Cu²⁺-S1P binding was bivalent and sensitive to the concentration of S1P in the SLB. In fact, the apparent equilibrium dissociation constant, KDApp, tightened by a factor of 132 from 4.5 μM to 34 nM as the S1P density was increased from 5.0 to 20 mol %. A major driving force for this apparent tightening was the more negative surface potential with increasing S1P concentration. This potential remained unaltered upon Cu²⁺ binding at pH 7.4 because two protons were released for every Cu²⁺ that bound. At pH 5.4, however, Cu²⁺ could not outcompete protons for the amine and no binding occurred. Moreover, at pH 9.4, the amine was partially deprotonated before Cu²⁺ binding and the surface potential became more positive on binding. The results for Cu²⁺-S1P binding were reminiscent of those for Cu²⁺-phosphatidylserine binding, where a carboxylate group helped to deprotonate the amine. In the case of S1P, however, the phosphate needed to bear two negative charges to facilitate amine deprotonation in the presence of Cu²⁺.
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In vitro brain tissue preparations allow the convenient and affordable study of brain networks and have allowed us to garner molecular, cellular, and electrophysiological insights into brain function with a detail not achievable in vivo. Preparations from both rodent and human post‐surgical tissue have been utilized to generate in vitro electrical activity similar to electrographic activity seen in patients with epilepsy. A great deal of knowledge about how brain networks generate various forms of epileptiform activity has been gained, but due to the multiple in vitro models and manipulations used, there is a need for a standardization across studies. Here, we describe epileptiform patterns generated using in vitro brain preparations, focusing on issues and best practices pertaining to recording, reporting, and interpretation of the electrophysiological patterns observed. We also discuss criteria for defining in vitro seizure‐like patterns (i.e., ictal) and interictal discharges. Unifying terminologies and definitions are proposed. We suggest a set of best practices for reporting in vitro studies to favor both efficient across‐lab comparisons and translation to in vivo models and human studies. This article is protected by copyright. All rights reserved.
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ELife digest Living cells have a surrounding membrane that largely insulates the space inside from that outside. Yet signals from outside of the cell can still influence how that cell behaves. One way this can happen is via small molecules called ligands binding to proteins embedded in the membrane and triggering a cascade of reactions inside the cell. Studying this kind of protein-ligand interaction is important for many aspects of biology. However, such studies typically require that the ligand be first labeled in some way, which is time-consuming, costly and may alter how the ligand behaves. As such, there is a need for alternative ways to measure ligands binding to proteins in membranes. The fact that the two sides of a membrane are insulated from each other means that they can store electrical charges. The ability to store charges is called capacitance. Theory predicts that it should be possible to detect a change in capacitance when a ligand binds to a membrane protein. Yet, though the theoretical basis of this hypothesis has been widely accepted, it had not been tested experimentally until now. Burtscher et al. chose to focus on a membrane protein called the serotonin transporter because a large number of its ligands had already been characterized. Experiments with human cells that expressed this transporter confirmed that the binding of a ligand was indeed detectable as a change in membrane capacitance. Burtscher et al. also detected a brief electrical current across the membrane that is predicted to occur when the capacitance changes. Ligand binding studies are especially important in therapeutics, as many drugs rely on blocking specific signaling pathways in diseased cells. As these capacitance recordings show precise real-time measurements, they could be used for drug screening in the future, all without the need to label the ligands.
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Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.
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1) A monolayer of lipids from bovine tongue epithelium was prepared as a model system for the taste receptor membrane and the influences of anion species on the surface potential were examined. The anion influence observed with the monolayer was closely correlated with that obtained with taste reception of the frog.2) The suppressive effect of salts on the response of the frog to sugars was examined by measuring activities of taste nerve. The order of effectiveness of the suppression was NaCl, KCl<MgCl2<MgSO4<K4 [Fe (CN) 6]. Plots of the response against the ionic strength fell on a single curve, which implied that the sugar response of the frog was suppressed by an increase of the ionic strength.3) The lipid monolayer penetrated with urease was prepared as a model system for a membrane containing a specific receptor protein. The change in the surface potential of the monolayer was observed with variation of concentration of urea or thiourea. No change in the surface potential was observed with use of inactive urease. It was inferred that the binding of the substrate to the enzyme induced a conformational change of the lipid membrane, which brought about a change in the surface potential.4) The fluorescence intensity of ANS in liposome suspension and the zeta-potential of he 1 iposome were measured as a function of concentration of various salts. It was concluded that an enhancement of fluorescence intensity is stemming from the changes in the surface potential of the liposome.5) Based on the analysis of the experimental results described above, discussion was made on the significance of the phase boundary potential in the membrane potential of biological systems.
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A theoretical treatment in terms of a carrier model is made to obtain a generalized equation for the BLM conductance produced by the charged complex formation between plural multivalent cations and plural charged carriers.The following experimental observations have been performed to deduce the ion transport mechanism across a BLM doped with Br-derivative of ionophore X537A (X-) according to the above-stated carrier model.The membrane conductance remarkably increases on the addition of X- to the aqueous solutions containing alkali metal cations.The membrane potential under a gradient of the concentration of monoor divalent cation shows quasi-Nernst behavior for the mono- or divalent cation, respectively.The ion conductance is proportional to approximately the square of the X- concentration, and it increases linearly with an increase in the ionic activities with a slope of 2 or 1 for the mono- or divalent cations, respectively, on a log-log plot.Thus it is concluded that the HX2- complex acts as a carrier for two alkali monovalent cations (I-) leading to a complex of I2HX2, and this carrier complex combines with one alkali divalent cation (12+) as well as with one hydrogen ion (H+) leading to a complex of IH2X22+.
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There is very little information about the spatial distribution of conductivity in nanopores. We calculated linearized conductivity from potential and current changes estimated by Poisson–Nernst–Planck and Navier–Stokes simulations of ion transport. The conductivity changes radially and axially. It is elevated near the wall of charged pores (or uncharged pores in the presence of electric field) with homogeneous diffusion within the nanopore, but is depressed if diffusion is slower near the wall. In unipolar nanopores, it is high in charged and uncharged half (regular bias), or moderate in the charged and low in uncharged half (reverse bias). In bipolar nanopores, the conductivity is high in both halves (but dips in the middle near the wall; regular bias). With reverse bias, it is very low throughout the nanopore, but the 3D resistivity plots reveal the resistivity to be much higher in the middle of the nanopore, critically influencing the pore resistance. Its restricted axial spread demonstrates that bipolar cylindrical nanofluidic diodes can be highly miniaturized. Graphical Abstract Both pore wall charge distribution and voltage bias affect the total conductivity of the nanopore, and its spatial distribution. As shown in unipolar nanopores the conductivity is high in charged and uncharged half (regular bias), or moderate in the charged and low in uncharged half (reverse bias) with homogeneous diffusion, but diminishes near the wall if diffusion becomes inhomogeneous, especially in the uncharged half (not shown). In bipolar nanopores, the conductivity is high within the nanopore, but dips near the wall in the middle, i.e., where the charges of opposite polarity meet (regular bias) with homogeneous diffusion, but decreases near the wall if diffusion becomes inhomogeneous (not shown). If the bias is reverse, the conductivity is very low throughout the nanopore irrespective of whether diffusion is homogeneous or inhomogeneous.
Chapter
Many important biological concepts follow from the ordering of biological space into intracellular and extracellular compartments as the plasma membrane is more than a structural boundary to contain the materials required for internal functions. On its outer surface the membrane bears molecules which receive a multitude of signals from the extracellular space. Laced through its phospholipid structure are other molecules which carry signals initiated by specific binding at receptor sites, and penetrating the membrane there are channel-forming molecules, some of which permit selective ion movement. Facing the cytoplasm, the inner leaflet is no less significant as it holds molecules which are the first participants in the steps which regulate cell biochemical functions via the second messenger systems.
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The voltage dependence of the voltage clamp responses of myelinated nerve fibers depends on the concentration of divalent cations and of hydrogen ions in the bathing medium. In general, increases of the [Ca], [Ni], or [H] increase the depolarization needed to elicit a given response of the nerve. An e-fold increase of the [Ca] produces the following shifts of the voltage dependence of the parameters in the Hodgkin-Huxley model: m∞, 8.7 mv; h∞, 6.5 mv; τn, 0.0 mv. The same increase of the [H], if done below pH 5.5, produces the following shifts: m∞, 13.5 mv; h∞, 13.5 mv; τn, 13.5 mv; and if done above pH 5.5: m∞, 1.3 mv; h∞, 1.3 mv; τn, 4.0 mv. The voltage shifts are proportional to the logarithm of the concentration of the divalent ions and of the hydrogen ion. The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels. The charged groups are assumed to comprise several types, of varying affinity for divalent and hydrogen ions. The charges near the sodium channels differ from those near the potassium channels. As the pH is lowered below pH 6, the maximum sodium conductance decreases quickly and reversibly in a manner that suggests that the protonation of an acidic group with a pKa of 5.2 blocks individual sodium channels.
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The voltage dependence of the voltage clamp responses of myelinated nerve fibers depends on the concentration of divalent cations and of hydrogen ions in the bathing medium. In general, increases of the [Ca], [Ni], or [H] increase the depolarization needed to elicit a given response of the nerve. An e-fold increase of the [Ca] produces the following shifts of the voltage dependence of the parameters in the Hodgkin-Huxley model: m(infinity), 8.7 mv; h(infinity), 6.5 mv; tau(n), 0.0 mv. The same increase of the [H], if done below pH 5.5, produces the following shifts: m(infinity), 13.5 mv; h(infinity), 13.5 mv; tau(n), 13.5 mv; and if done above pH 5.5: m(infinity), 1.3 mv; h(infinity), 1.3 mv; tau(n), 4.0 mv. The voltage shifts are proportional to the logarithm of the concentration of the divalent ions and of the hydrogen ion. The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels. The charged groups are assumed to comprise several types, of varying affinity for divalent and hydrogen ions. The charges near the sodium channels differ from those near the potassium channels. As the pH is lowered below pH 6, the maximum sodium conductance decreases quickly and reversibly in a manner that suggests that the protonation of an acidic group with a pK(a) of 5.2 blocks individual sodium channels.
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(1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs+ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs+>Rb+>K+>Na+>Li+. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm −1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.
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This paper, the last in a series of three, characterizes the electrical properties of phospholipid bilayer membranes exposed to aqueous solutions containing nonactin, monactin, dinactin, and trinactin and Li+, Na+, K+, Rb+, Cs+, and NH 4+ ions. Not only are both the membrane resistance at zero current and the membrane potential at zero current found to depend on the aqueous concentrations of antibiotic and ions in the manner expected from the theory of the first paper, but also these measurements are demonstrated to be related to each other in the manner required by this theory for “neutral carriers”. To verify that these antibiotics indeed are free to move as carriers of cations, cholesterol was added to the lipid to increase the “viscosity” of the interior of the membrane. Cholesterol decreased by several orders of magnitude the ability of the macrotetralide antibiotics to lower the membrane resistance; nevertheless, the permeability ratios and conductance ratios remained exactly the same as in cholesterolfree membranes. These findings are expected for the “carrier” mechanism postulated in the first paper and serve to verify it. Lastly, the observed effects of nonactin, monactin, dinactin, and trinactin on bilayers are compared with those predicted in the preceding paper from the salt-extraction equilibrium constants measured there; and a close agreement is found. These results show that the theory of the first paper satisfactorily predicts the effects of the macrotetralide actin antibiotics on the electrical properties of phospholipid bilayer membranes, using only the thermodynamic constants measured in the second paper. It therefore seems reasonable to conclude that these antibiotics produce their characteristic effects on membranes by solubilizing cations therein as mobile positively charged complexes.
Article
In order to clarify the mechanism by which neutral molecules such as the macrotetralide actin antibiotics make phospholipid bilayer membranes selectively permeable to cations, we have studied, both theoretically and experimentally, the extraction by these antibiotics of cations from aqueous solutions into organic solvents. The experiments involve merely shaking an organic solvent phase containing the antibiotic with aqueous solutions containing various cationic salts of a lipid-soluble colored anion. The intensity of color of the organic phase is then measured spectrophotometrically to indicate how much salt has been extracted. From such measurements of the equilibrium extraction of picrate and dinitrophenolate salts of Li, Na, K, Rb, Cs, and NH4 into n-hexane, dichloromethane, and hexane-dichloromethane mixtures, we have verified that the chemical reactions are as simple as previously postulated, at least for nonactin, monactin, dinactin, and trinactin. The equilibrium constant for the extraction of each cation by a given macrotetralide actin antibiotic was also found to be measurable with sufficient precision for meaningful differences among the members of this series of antibiotics to be detected. It is noteworthy that the ratios of selectivities among the various cations were discovered to be characteristic of a given antibiotic and to be completely independent of the solvent used. This finding and others reported here indicate that the size and shape of the complex formed between the macrotetralide and a given cation is the same, regardless of the species of cation bound. For such "isosteric" complexes, notable simplifications of the theory become possible which enable us to predict not only the electrical properties of a membrane made of the same solvent and having the thinness of the phospholipid bilayer but also, and more importantly, the electrical properties of the phospholipid bilayer membrane itself. These predictions will be compared with experimental data for phospholipid bilayer membranes in the accompanying paper.
Article
A smectic mesophase (myelin-like structure, layer-latticed liquid crystal) of charged phospholipid behaves as an almost perfect osmometer when alkali metal salts, glucose, sucrose or mannitol are used as solutes. Other solutes show graded permeabilities (ethylurea, methylurea, ethylene glycol, ammonium acetate, propionamide, glycerol > urea > malonamide > erythritol). Osmotically driven swelling and shrinkage were followed by means of the changes in optical extinction; the validity of this was confirmed by determination of the volumes and interstitial spaces of centrifuged pellets. The rapid volume changes, determined optically, combined with the measured total external surface areas of the phospholipid dispersions, were used to calculate osmotic water permeability coefficients (0.8–16 μsec−1).The complementary effects of surface charge and electrolyte concentration on the equilibrium volumes of smectic mesophases were examined optically and by centrifugation. The volume of the particles decreased with increasing concentration of the electrolyte solution in which they formed. The intramellar spacings were not consistent with a single Hamaker constant over the range of electrolyte concentration and area-charge ratio studied. The constant was high (10-11 ergs) with low electrolyte concentrations and lower by a factor of about 100 with a high electrolyte concentration.
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The results of present experiments demonstrate the stabilizing action of Ca, Mg, Sr and Ba, and suggest that Ca ions contribute little to the inward membrane ionic current during the rising phase of the action potential in the cockroach giant axons.
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We present and discuss the permeability and electrical properties of thin lipid membranes, and the changes induced in these properties by several agents added to the aqueous phases after the membranes have formed. The unmodified membrane is virtually impermeable to ions and small "hydrophilic" solutes, but relatively permeable to water and "lipophilic" molecules. These properties are consistent with those predicted for a thin film of hydrocarbon through which matter is transported by dissolving in the membrane phase and then diffusing through it. The effect of cholesterol in reducing the water and "lipophilic" solute permeability is attributed to an increase of the "viscosity" of the hydrocarbon region, thus reducing the diffusion coefficient of molecules within this phase. The selective permeability of the membrane to iodide (I(-)) in the presence of iodine (I(2)) is attributed to the formation of polyiodides (perhaps I(5) (-)), which are presumed to be relatively soluble in the membrane because of their large size, and hence lower surface charge density. Thus, I(2) acts as a carrier for I(-). The effects of "excitability-inducing material" and the depsipeptides (particularly valinomycin) on ion permeability are reviewed. The effects of the polyene antibiotics (nystatin and amphotericin B) on ion permeability, discussed in greater detail, are the following: (a) membrane conductance increases with the 10th power of nystatin concentration; (b) the membrane is anion-selective but does not discriminate completely between anions and cations; (c) the membrane discriminates among anions on the basis of size; (d) membrane conductance decreases extraordinarily with increasing temperatures. Valinomycin and nystatin form independent conductance pathways in the same membrane, and, in the presence of both, the membrane can be reversibly shifted between a cation and anion permeable state by changes in temperature. It is suggested that nystatin produces pores in the membrane and valinomycin acts as a carrier.
Article
1. 1. Two membrane fractions were isolated from homogenates of the first stellar nerve of Dosidicus gigas by a sequence of differential, discontinuous and gradient centrifugations in sucrose solutions. Membrane Fraction I has an apparent density of 1.090 g/ml and membrane Fraction II a value of 1.140 g/ml. 2. 2. At low and high magnifications in the electron microscope, both fractions appear as rounded membrane profiles similar to the plasma membrane observed in the unfractionated tissue. No other subcellular component was observed. Fraction I has a thickness of 95-110 Å and that of Fraction II is 72-100 Å. 3. 3. The percentage lipid composition of the two membranes was established. The following lipid classes were tentatively identified: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, cholesterol, free fatty acids and the hydrocarbon n-pentacosane. The relative distribution of the fatty acids present in the phospholipids of the two membrane fractions was different. 4. 4. The morphological appearance, the yield of the two membranes and the distribution of (Na+-K+)-dependent, ouabain-sensitive ATPase has led us tentatively to relate Fraction I with the axolemma and Fraction II with the Schwann cell plasma membrane.
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Clear dispersions of 8-10 mg of ammonium phosphatidylinositol or sodium phosphatidylinositol in 5 ml of water obtained by gentle sonic radiation were titrated in the pH range of 2.5-10.0. The initial pH was 6.1-6.5. The pK′ of phosphatidylinositol, which was 3.12 in the absence of electrolyte, was lowered to 2.50 in 0.08 M NaCl. The interaction of phosphatidylinositol with metallic cations was studied by three methods. (1) Hydrogen ion release: Dispersions of phosphatidylinositol were adjusted to pH 3.5 and increments of electrolyte were added. The quantity of H+ released (measured by the quantity of tetramethylammonium hydroxide required to maintain pH 3.5) followed the order Ca2+ > Mg2+ ≫ K+ > Na+. (2) Turbidity: The turbidity of dispersions was measured by the scattering ratio I90°/I0°. The effectiveness of the cations in increasing turbidity was Ca2+ > Mg2+ ≫ K+ > Na+ > Li+ > choline chloride. (3) Coagulation: Dispersions of phosphatidylinositol were coagulated by the addition of salts. Analysis of cations in the coagula provided a measure of cation selectivity. The molar ratio of divalent cation to phosphatidylinositol of 0.5 indicated bridging of two phosphatidylinositol molecules by each Ca2+ or Mg2+. The selectivity ratios were Ca2+ : Mg2+ ≃ 2.4 and K+ : Na+ ≃ 1.2. An apparent constant for the association of sodium phosphatidylinositol was obtained from data on cation-proton exchange at pH 3.5, KNa′ = 6.9. The viscosity of phosphatidylinositol dispersions in water was much greater than for other acidic lipids; however, a low concentration (2.5 mM) of NaCl produced a sharp decrease.
Article
The interaction of phospholipids with bivalent metals was studied by the use of monolayers and liquid crystals composed of purified, naturally occurring phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid and phosphatidylinositol. The properties under investigation were surface pressure, surface potential and ξ potential. Changes in these parameters were followed as a function of pH and bivalent metal ion concentration.It was found that the acidic phospholipids interact strongly with bivalent metals at low concentrations (10−4−10−3 M), in the presence of physiological concentrations of univalent salts (10−1 M). The interaction is accompanied by an increase in surface potential and a decrease in surface pressure (condensation) of monomolecular films. Of the phospholipids examined, phosphatidylserine and phosphatidic acid at pH values higher than 7.0, both possessing two negative groups per molecule, exhibited the highest affinity for bivalent metals.On the basis of the results presented, a model was proposed for the complex of the latter two phospholipids with Ca2+. The model involves coordination bonds between each Ca2+ and four phospholipid molecules resulting in a linear polymeric arrangement. The implications of the complex were discussed in terms of the physiological role of bivalent metals on membranes.
Article
The fluorescence of 8-anilino-1-naphthalene sulfonate (ANS)3 is enhanced by skeletal muscle microsomes and micellar dispersions of phospholipids. The magnitude of the enhancement is a unique function of the ionic composition, rising with the concentration of cations according to a titration curve. The effect of cations might reflect the increased hydrophobic character of the membrane or the increased binding of ANS, induced by cations. The optimum pH for the ANS fluorescence in the presence of microsomes or lecithin is at pH 1–4 and at pH 7.3 a complex temperature dependence was observed with indications of a transition at 35–40 °. Treatment of microsomes or lecithin with phospholipase C causes a decrease of ANS fluorescence while trypsin digestion had little or no effect. Polymyxin B, a circular polypeptide, inhibits the ATPase activity and Ca2+ transport of muscle microsomes accompanied by a large increase in fluorescence enhancement in the presence of ANS. Tyrocidine also inhibited the biochemical functions while gramicidin was less effective. Polyene antibiotics were generally without effect.These observations indicate that caution is required in the evalution of ANS fluorescence data in terms of conformational changes in membrane proteins alone, since the contribution of phospholipids to the fluorescence is significant.
Article
In summary a wide variety of experiments with axons provides kinetic, electrochemical, and pharmacological evidence that three types of channels through the membrane contribute to the ionic permeability changes underlying action potentials. In normal function one channel accounts for most of the movements of Na ions, another accounts for most of the movements of K ions, and the last accounts for the remaining “leakage” fluxes.The Na, K, and leakage channels seem to be independent specializations of the membrane. Theoretical, electrical, and pharmacological evidence suggest that Na channels are short narrow pores that open and close in an all-or-nothing fashion. An open Na channel in a squid giant axon may have a conductance of about 0.5 nmho. Very little is known about K and leakage channels except that K channels may be longer pores with a lower conductance than Na channels.
Article
Bilayer membranes, formed from various phospholipids, were studied to assess the influence of the charge of the polar head groups on the membrane conductance mediated by neutral "carriers" of cations and anions. The surface charge of an amphoteric lipid, phosphatidyl ethanolamine, was altered by varying the pH, and the surface charge of several lipids was screened by increasing the ionic strength of the solution with impermeant monovalent and divalent electrolytes. The surface charge should be a key parameter in defining the membrane conductance for a variety of permeation mechanisms; conductance measurements in the presence of carriers may be used to estimate the potential difference, due to surface charge, between the interior of the bilayer and the bulk aqueous phase. The large changes in conductance observed upon varying the surface charge density and the ionic strength agree with those predicted by the Gouy-Chapman theory for an aqueous diffuse double layer. Explicit expressions for the dependence of the membrane conductance on the concentrations of the carrier, the permeant ion, the surface charge density, and the ionic strength are presented.
Article
THE principal permeability barrier in biological membranes seems to be a layer of hydrocarbon chains in a bilayer of phospholipid molecules1. We now report studies on the physical state of this hydrocarbon layer; this relates to the permeability of membranes2. Early X-ray diffraction studies of membranes showed that the hydrocarbon chains in membranes are not in a crystalline array3. The marked similarity between the rather broad X-ray diffraction band at 4.6 Â obtained from membranes and that derived from liquid paraffins shows that the hydrocarbon chains in membranes are in a liquid state.The 4.6 Â diffraction from liquid paraffins is at right angles to the chain length and is correlated with the interchain distances4,5. The 4.6 Â band from myelinated nerve3 is oriented at right angles to the low angle reflexions. There is little doubt that this diffraction is caused largely by scattering from the hydrocarbon chains, but the deduction3 that the chains are oriented perpendicular to the plane of the myelin membrane is somewhat uncertain, because the 4.6 Â diffraction band probably contains contributions from scattering from membrane protein.
Article
Summary In this paper we derive expressions for the ion flux across lipid bilayer membranes with charged surfaces treating the membrane as a continuous phase interposed between two electrolyte solutions and calculating the ion flux with the Nernst-Planck equations. The theoretical results are compared with experiments of Seufert and Hashimoto on lipid bilayer membranes with charged surface active agents added to the membranes. If the charge of both membrane surfaces has the same sign the flux of the gegenions is greatly increased whereas the flux of the coions decreases to a small amount. For oppositely charged membrane surfaces the membrane behaves like a np semiconductor and typical rectification voltage-current characteristics are obtained.
Article
THE curve relating membrane conductance (Gm) to membrane potential (Vm) shifts along the voltage axis with changes in concentration of external divalent ions1-4, external pH5 and internal ionic strength6. These findings are in qualitative agreement with the suggestion made by Huxley1 that the shift may correspond to changes in the electric field resulting from changes in the membrane surface potential, ψ0. To test the validity of the suggestion experimental shifts need to be compared with calculated changes in ψ0 when ψ0 is affected by various factors. While calculating ψ0 it is essential to use the same basic assumptions concerning the charge number and chemical nature of adsorbing groups (binding sites) on the membrane surface. To this end, we studied the effect of changes in the external concentrations of H+, Ca2+, Mg2+, Co2+, Ni2+ and external ionic strength on steadystate potassium conductance (Gm-Vm curves).
Article
1. The effect of extracellular calcium and magnesium on the contraction threshold and on the thresholds for an increase in sodium and potassium conductance with depolarization was studied in voltage-clamped frog muscle fibres.2. A larger depolarization was required to reach each of the three thresholds when the concentration of divalent cation was increased.3. The contraction and potassium conductance thresholds appeared to shift in parallel with alterations in calcium over the concentration range 0.2-10.0 mM and in magnesium over the concentration range 5.4-90.0 mM. The shift amounted to about 4 mV for a threefold change in concentration of divalent cation.4. The sodium conductance threshold was much more sensitive to alterations in divalent cation concentration than was either the contraction or the potassium conductance threshold.
Article
Calcium appears to be an essential participant in axon excitation processes. Many other polyvalent metal ions have calcium-like actions on axons. We have used the voltage-clamped lobster giant axon to test the effect of several of these cations on the position of the peak initial (sodium) and steady-state (potassium) conductance vs. voltage curves on the voltage axis as well as on the rate parameters for excitation processes. Among the alkaline earth metals, Mg(+2) is a very poor substitute for Ca(+2), while Ba(+2) behaves like "high calcium" when substituted for Ca(+2) on a mole-for-mole basis. The transition metal ions, Ni(+2), Co(+2), and Cd(+2) also act like high calcium when substituted mole-for-mole. Among the trivalent ions, La(+3) is a very effective Ca(+2) replacement. Al(+3) and Fe(+3) are extremely active and seem to have some similar effects. Al(+3) is effective at concentrations as low as 10(-5)M. The data suggest that many of these ions may interact with the same cation-binding sites on the axon membrane, and that the relative effects on the membrane conductance and rate parameters depend on the relative binding constants of the ions. The total amount of Na(+) transferred during a large depolarizing transient is nearly independent of the kind or amount of polyvalent ion applied.
Article
Article
Potassium conductance-voltage curves have been determined for a squid axon in high external potassium solution for a wide range of divalent cation concentrations. A decrease in divalent ion concentration shifts the conductance-voltage curve along the voltage axis in the direction of more hyperpolarized voltages by as much as 9 mv for an e-fold change in concentration. When the divalent ion concentration is less than about 5 mM, a further decrease does not cause a significant shift of the conductance-voltage curve. These results can be explained by assuming that on the outer surface of the membrane there is a negative fixed charge which can bind calcium ions, and that the axon is sensitive to the resulting double-layer potential. From our data, the best value for charge density was found to be one electronic charge per 120 square angstroms, and a lower limit to be one electronic charge per 280 square angstroms.
Article
In studying the functions of phosphatides in biological membranes, investigations have been made into monomolecular layers of synthetic phosphatides. Force-area curves of monolayers of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, all containing identical fatty acids, showed small differences, obviously to be attributed to differences in size and charge of the end groups. The shifts of the force-area curves within one class of phosphatides were more pronounced and are brought about by variations of the apolar moiety. Shortening of the chain length and particularly unsaturation of the fatty acid constituents greatly expanded the films of the l-α-lecithins, thereby increasing the closest stable packing attainable. Force-area curves of structurally isomeric mixed-acid l-α-phosphatides, carrying dissimilar fatty acids in different positions, were identical. Mixed films consisting of phosphatides and cholesterol in molar equivalents—at proportions also found in red cell membranes—revealed a condensing effect of cholesterol on the film of phosphatides containing certain unsaturated fatty acid constituents.
Interfacial Phenomena
• E K Rideal
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b. Ionic properties of phosphatidic acid
• M B Abramson
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Location of l-anilino-8-napthalene-sulfonate (ANS) in lipid bimolecular leaflets determined by direct analysis of X-ray diffraction data
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Competition between Ca and H ions at the nerve surface
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