Voltage-induced gating of the mechanosensitive MscL ion channel reconstituted in a tethered lipid bilayer membrane

The mechanosensitive (MS) ion channel is gated by changes in bilayer deformation. It is functional without the presence of any other proteins and gating of the channel has been successfully achieved using conventional patch clamping techniques where a voltage has been applied together with a pressure over the membrane. Here, we have for the first time analyzed the large conducting (MscL) channel in a supported membrane using only an external electrical field. This was made possible using a newly developed technique utilizing a tethered lipid bilayer membrane (tBLM), which is part of an engineered microelectronic array chip. Single ion channel activity characteristic for MscL was obtained, albeit with lower conductivity. The ion channel was gated using solely a transmembrane potential of 300 mV. Computations demonstrate that this amount of membrane potential induces a membrane tension of 12 dyn/cm, equivalent to that calculated to gate the channel in patch clamp from pressure-induced stretching of the bilayer. These results strengthen the supposition that the MscL ion channel gates in response to stress in the lipid membrane rather than pressure across it. Furthermore, these findings illustrate the possibility of using the MscL as a release valve for engineered membrane devices; one step closer to mimicking the true function of the living cell.     

Modification of latrotoxin channels under the effect of pronase and antibodies to latrotoxin

The action of pronase and latrotoxin antibodies on alpha-latrotoxin channels in lipid bilayers has been investigated. Proteolysis of the protruding portions of the inserted channels changed slightly its conductivity and selectivity. Latrotoxin antibodies added to either side of the membrane decreased channel conductivity. The effect depends on the membrane potential. It is suggested that the channel conformation depends on the transmembrane potential and selective centre localized in the inner region of a bilayer is insensitive to proteolysis of protruding segments.     

Glycoproteins bound to ion channels mediate detection of electric fields: a proposed mechanism and supporting evidence

The mechanism by which animals detect weak electric and magnetic fields has not yet been elucidated. We propose that transduction of an electric field (E) occurs at the apical membrane of a specialized cell as a consequence of an interaction between the field and glycoproteins bound to the gates of ion channels. According to the model, a glycoprotein mass (M) could control the gates of ion channels, where M > 1.4 x 10(-18)/E, resulting in a signal of sufficient strength to overcome thermal noise. Using the electroreceptor organ of Kryptopterus as a mathematical and experimental model, we showed that at the frequency of maximum sensitivity (10 Hz), fields as low as 2 microV/m could be detected, and that the observation could be explained if a glycoprotein mass of 0.7 x 10(-12) kg (a sphere 11 microm in diameter) were bound to channel gates. Antibodies against apical membrane structures in Kryptopterus blocked field transduction, which was consistent with the proposal that it occurred at the membrane surface. Although the target of the field was hypothesized to be an ion channel, the proposed mechanism can easily be extended to include other kinds of membrane proteins.     

Kinetics of channelized membrane ions in magnetic fields

The cyclotron resonance model for channel ion transport in weak magnetic fields is extended to include damping losses. The conductivity tensor is obtained for different electric field configurations, including the circuital field E phi normal to the channel axis. The conductivity behavior close to the cyclotron resonance frequency omega c is compared to existing Ca2+-efflux data in the literature. A collision time of .023 s results from this comparison under the assumption that K+ ions are transiting in a 0.35 G field. We estimate a mean kinetic energy of 3.5 eV for this ion at resonance. This model leads to discrete modes of vibration (eigenfrequencies) in the ion-lattice interaction, such that omega n = n omega c. The presence of such harmonics is compatible with recent results by Blackman et al. [1985b] and McLeod et al. [1986] with the interesting exception that even modes do not appear in their observations, whereas the present model has no restriction on n. This harmonic formalism is also consistent with another reported phenomenon, that of quantized multiple conductances in single patch-clamped channels.     

Single calcium-dependent potassium channels in clonal anterior pituitary cells.

Single Ca2+-dependent K+-channel currents were recorded in intact and excised inside-out membrane patches of the anterior pituitary clone AtT-20/D16-16. The frequency of channel openings and lifetimes depends both on membrane potential and on the Ca2+ concentrations at the inner membrane surface. The curve of the open-state probability of the channel as a function of membrane potential appears to translate along the voltage axis with changes in internal Ca2+ concentration. For Ca2+ concentrations between 10(-7) and 10(-6) M, the shift is consistent with the hypothesis that three Ca2+ ions are required to open a Ca2+-dependent K+ channel. Single channel conductances are estimated to be 124 pS in patches with normal external K+ (5.4 mM) and 208 pS in excised patches with symmetrical K+ (145 mM) across the membrane. Tetraethylammonium (20 mM) added to the cytoplasmic surface reversibly blocks the Ca2+-dependent K+ channel.     

Voltage dependence of subthreshold resonance frequency in layer II of medial entorhinal cortex

The resonance properties of individual neurons in entorhinal cortex (EC) may contribute to their functional properties in awake, behaving rats. Models propose that entorhinal grid cells could arise from shifts in the intrinsic frequency of neurons caused by changes in membrane potential owing to depolarizing input from neurons coding velocity. To test for potential changes in intrinsic frequency, we measured the resonance properties of neurons at different membrane potentials in neurons in medial and lateral EC. In medial entorhinal neurons, the resonant frequency of individual neurons decreased in a linear manner as the membrane potential was depolarized between -70 and -55 mV. At more hyperpolarized membrane potentials, cells asymptotically approached a maximum resonance frequency. Consistent with the previous studies, near resting potential, the cells of the medial EC possessed a decreasing gradient of resonance frequency along the dorsal to ventral axis, and cells of the lateral EC lacked resonant properties, regardless of membrane potential or position along the medial to lateral axis within lateral EC. Application of 10 μM ZD7288, the H-channel blocker, abolished all resonant properties in MEC cells, and resulted in physiological properties very similar to lateral EC cells. These results on resonant properties show a clear change in frequency response with depolarization that could contribute to the generation of grid cell firing properties in the medial EC.     

Proton exclusion by an aquaglyceroprotein: a voltage clamp study

BACKGROUND INFORMATION: In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers. RESULTS: To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7x10(-14) cm(3).subunit(-1).s(-1). Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H(+) (OH(-)) to H(2)O molecules transport stoichiometry of 2x10(-9). A significant GlpF-mediated ion conductivity was also not detectable. CONCLUSIONS: The lack of a physiologically relevant GlpF-mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.     

Activity of the plant peptide aglycin in mammalian systems

A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27-63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 microg.g(-1) body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

Nerve membrane electrical characteristics near the resting state

Summary An experimental analysis of the squid axon membrane impedance in the vicinity of the resting state and as a function of frequency is presented. Particular attention was devoted to the measurement of theresonance frequency, for which the absolute magnitude of the impedance attains its maximum value, in different, extracellular solutions, at various temperatures and in the presence of constant depolarizations or hyperpolarizations.The variations in the concentration of sodium, potassium and divalent ions and the addition of tetrodotoxin, changed markedly the maximum impedance but had little effect, at a fixed temperature, on the resonance frequency, whose temperature dependance is described by aQ ₁₀ variable from 3.7 (around 4 °C) to 1.9 (around 15 °C). Substitution of heavy water decreased the resonance frequency by a factor 1.25, fairly independent of temperature. Steady depolarizations or hyperpolarizations produced large variations of the resonance frequency, with strong temperature dependance.The results indicate that the resonance frequency is directly related to the membrane permeability changes which take place quite independently of the composition of the extra cellular solution and are governed by the electric field existing within the membrane structure rather than by the total membrane potential, to which membrane-solution boundary potentials can give a large contribution.     

Phencyclidine and some of its analogues have distinct effects on NMDA receptors of rat hippocampal neurons

Phencyclidine (PCP) is a dissociative anesthetic agent which blocks the excitatory effect of N-methyl-D-aspartate (NMDA) in the central nervous system. To investigate the role of the PCP reactive site in the control of NMDA activation of hippocampal pyramidal cells, we have examined the action of PCP and some of its analogues on the response properties of single NMDA receptors. Application of NMDA (5-15 microM) to outside-out patches of membrane elicited bursts of ion channel openings which were greatly reduced in frequency and duration in the presence of PCP (2.5-10 microM) or m-amino-PCP (2.5-10 microM), a behaviorally active derivative of PCP. These effects of PCP were reversed when the membrane potential was shifted from negative to positive values. Application of the behaviorally inactive agent 1-piperidino-cyclohexanecarbonitrile (greater than or equal to 220 microM) left NMDA-activated currents relatively unaltered. Treatment with another analogue, m-nitro-PCP (5-20 microM), resulted in an unexpected increase in frequency of openings. At a higher concentration (100-300 microM), however, m-nitro-PCP acted like PCP in reducing frequency of opening and channel life-time. Like PCP, these effects of m-nitro-PCP were reversed at positive potentials. Taken together, these results suggest that PCP and its derivatives block the open state of the NMDA channel. Moreover, the dual effect of m-nitro-PCP shows that excitability is not necessarily decreased by PCP analogues but may instead be enhanced depending on modifications of the PCP molecule.     

Alteration of the passive electrical properties of lymphocyte membranes induced by GM1 and GM3 glycolipids.

The electrical conductivity of normal human lymphocyte suspensions has been measured in the frequency range from 10 kHz to 100 MHz, where a well-pronounced conductivity dispersion occurs, caused by the surface polarization at the interface between the cell membrane and the extracellular solution. We have investigated the alteration of the passive electrical properties of the cytoplasmatic cell membrane induced by two different gangliosides (GM1 and GM3) inserted, at various concentrations, into the outer leaflet of membrane double layer. The alterations observed in the dielectric parameters (the membrane conductivity and the membrane permittivity) derived on the basis of a 'double-shell' model, result in an overall increase of the ion permeation across the membrane and an enhanced polarizability of its hydrophilic region for both gangliosides investigated. The relevance of these alterations is discussed.     

resonant events in membranes having ion channels with two conformational states

A study was carried out of a mathematical model of ion transport through biological membranes along the channels capable of conformational transitions between two states (R, T) with different conductivities. The model describes changes in time of te membrane potential and surface concentration of channels in one of the states (R). It has been shown that there may exist extinguishing oscillations with the frequency close to f0 on such a system may induce the resonance changes of the membrane potential. The resonance frequency f0 increases with an increase of the amplitude of external influence, this induces hesteresis of the membrane resonance parameters.     

Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis

By using the patch-clamp technique, stretch-activated ionic channels were found in the membrane of cleaving freshwater fish embryos at the early stages of embryogenesis (2-256 cells). The application of negative pressure to the pipette increased the frequency of activation and the duration of bursts. This type of channel has a preferential K+ selectivity. When bathed on both membrane surfaces with 140 mM KCl the channel conductance was 71 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration on the cytoplasmic side of the membrane. On continuous recording, the probability of the channel being open was found to change periodically over a 5- to 20-fold range for different cells. These variations correlated with changes in resting potential and membrane conductance during the cell cycle. These results suggest that the oscillation of resting potential within the cell cycle is associated with the operation of stretch-activated ion channels.     

A novel method for measuring membrane conductance changes by a voltage-sensitive optical probe.

This study presents a method whose principles enable using a voltage-sensitive optical probe, to quantitatively measure conductivity changes elicited in membrane vesicles and cells. The procedure is based on the fact that the amplitude of the transmembrane potential difference, established across a membrane by an external electric field, is decreased when membrane conductivity is increased upon incorporation of ionophores into the membrane. The method was applied to osmotically swollen thylakoid membranes whose membrane conductivity was changed by the addition of gramicidin or ionomycin. The electric field induced stimulated luminescence from photosystem I (electrophotoluminescence-EPL) was used as a voltage-sensitive optical probe. We calculated the induced conductance changes by using a calibrated EPL vs external electric field response curve and measuring the ionophore-mediated attenuation of the EPL signal. The calculated ionophore-unmodified conductance of the thylakoid membrane yields a value of 171 +/- 56 nS/cm. The value of the membrane conductance, modified by 10 nM gramicidin was found to be 190 +/- 56 nS/cm. The modified membrane conductance and the membrane conductance changes induced by 1 microM ionomycin in the presence of CaCl2 were found to be 186 +/- 3 nS/cm and 15 +/- 3 nS/cm, respectively.     

Sensitivity of membranes to their environment. Role of stochastic processes

Ionic flow through biomembranes often exhibits a sensitivity to the environment, which is difficult to explain by classical theory, that usually assumes that the free energy available to change the membrane permeability results from the environmental change acting directly on the permeability control mechanism. This implies, for example, that a change delta V in the trans-membrane potential can produce a maximum free energy change, delta V X q, on a gate (control mechanism) carrying a charge q. The analysis presented here shows that when stochastic fluctuations are considered, under suitable conditions (gate cycle times rapid compared with the field relaxation time within a channel), the change in free energy is limited, not by the magnitude of the stimulus, but by the electrochemical potential difference across the membrane, which may be very much greater. Conformational channel gates probably relax more slowly than the field within the channel; this would preclude appreciable direct amplification of the stimulus. It is shown, however, that the effect of impermeable cations such as Ca++ is to restore the amplification of the stimulus through its interaction with the electric field. The analysis predicts that the effect of Ca++ should be primarily to affect the number of channels that are open, while only slightly affecting the conductivity of an open channel.     

Dielectric properties of E. coli cell as simulated by the three-shell spheroidal model

Dielectric properties of E. coli cell have been re-studied by means of the three-shell spheroidal model, where the three shells correspond to the outer membrane, the periplasmic space and the inner membrane, respectively. With the model, a curve-fitting procedure has been developed to analyze the dielectric spectra. Although E. coli cell has been studied before, its special morphological structure was taken into account more comprehensively than any previous model in the present work. Dielectric properties of various cell components have been estimated from the observed dielectric spectra, especially the permittivity of the outer membrane, which was evaluated quantitatively for the first time. The values of epsilon(om) were 12 for kappa(om) of 0 to 10(-4) S/m and 34 for kappa(om) of 10(-3) S/m. The specific capacitance of the inner membrane was 0.6-0.70 microF/cm(2). The relative permittivity and the conductivity of the cytoplasm were about 100 and 0.22 S/m, respectively, and the conductivity of the periplasmic space was 2.2-3.2 S/m.     

Parametric resonance and amplification in excitable membranes. The Hodgkin-Huxley model

It has previously been shown by different investigators that the excitable membrane shows a resonant sensitivity to periodic external perturbations, but its Q-factor is, as a rule, low. The present paper analyses the possible ways of increasing the membrane Q, using a model of the Hodgkin-Huxley type. It is found, in particular, that it can be increased considerably by modulating periodically the membrane capacitance or the activation and inactivation rate constants of ionic channels, with a frequency of about 2 fo (fo being the fundamental frequency of damped oscillations in the membrane), the extent of modulation not exceeding the critical value 2/Q. In this case, a significant parametric amplification of the membrane current takes place. If the modulation coefficient is above 2/Q, the membrane can display a parametric resonance that causes stable self-oscillations in the potential with a frequency approximately fo. The conditions for the realization of parametric amplification and resonance in biological membranes are discussed.     

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells.     

Regulation of potassium conductance in the cellular membrane at early embryogenesis.

At the early stages of development of the fresh water fish loach (Misgurnus fossilis) the resting membrane potential (Er) of cleaving cells oscillates periodically with an amplitude of 8-12 mV. Er oscillation correlates with the cell cycle and is accompanied by changes of K+ conductivity. Two types of K(+)-selective ionic channels with conductance of approximately 70 and 25 pS in symmetrical (150 mM KCl) solution were observed in the membrane of cleaving loach embryos. 'High' conductance and 'low' conductance channels were recorded in approximately 90% and 10% of patches investigated (n = 275), respectively? The activity of 'high' conductance channels was regulated by the application of pressure to the membrane, ie these channels were stretch-activated (SA). The activity of SA channels changes dramatically during the cell-cleavage cycle. At the beginning of interphase the probability of SA channels being in the open state (P0) was minimal, while at prometaphase the probability was increased 10-100-fold. Application of ATP to the cytoplasmic inside-out patches induced a reversible elevation of stretch sensitivity of the SA channels in 50% of the patches, while the non-hydrolyzable analogue of ATP was not effective. Combined application of ATP, cAMP and cAMP-dependent protein kinase (PK) induced a reversible elevation in the SA channel activity while inhibitors of PK prevented its activating effects. Phosphatase inhibitors prolonged the activating effect of PK on SA channels. We propose that oscillations of the resting potential during the cell-cleavage cycle arise due to modulation of SA channel sensitivity to stretch through cAMP-dependent phosphorylation.