Fluid domain patterns in free-standing membranes captured on a solid support

We devise a methodology to fixate and image dynamic fluid domain patterns of giant unilamellar vesicles (GUVs) at sub-optical length scales. Individual GUVs are rapidly transferred to a solid support forming planar bilayer patches. These are taken to represent a fixated state of the free standing membrane, where lateral domain structures are kinetically trapped. High-resolution images of domain patterns in the liquid-ordered (l_o) and liquid-disordered (l_d) co-existence region in the phase-diagram of ternary lipid mixtures are revealed by atomic force microscopy (AFM) scans of the patches. Macroscopic phase separation as known from fluorescence images is found, but with superimposed fluctuations in the form of nanoscale domains of the l_o and l_d phases. The size of the fluctuating domains increases as the composition approaches the critical point, but with the enhanced spatial resolution, such fluctuations are detected even deep in the coexistence region. Agreement between the area-fraction of domains in GUVs and the patches respectively, supports the assumption that the thermodynamic state of the membrane remains stable. The approach is not limited to specific lipid compositions, but could potentially help uncover lateral structures in highly complex membranes.     

The elastic deformability of closed multilayered membranes is the same as that of a bilayer membrane

The elastic behavior of closed multilayered membranes is analyzed with the assumption that the constituent layers are in close contact but are unconnected in the sense that they are free to slide by one another. The system exhibits three independent elastic deformation modes for any number of the constituent layers equal to or larger than two. These are the area expansivity of the membrane neutral surface, and the local and non-local membrane bending. The corresponding elastic moduli are expressed in terms of the elastic moduli of the constituent layers, their areas, and distances between their neutral surfaces. Closed multilayered membranes only differ from a closed bilayer membrane in that for any of their shapes some of the constituent layers are expanded and some compressed.     

The roles of intracellular regions in the activation of voltage-dependent potassium channels

The involvement of the transmembrane regions S2, S3 and S4 in the activation of potassium channels by depolarization has been well clarified. However, a role of the intracellular regions in channel function is emerging. Here we review recent evidence for the roles of intracellular regions in the functioning of members of two families of channels. The Kv2.1 potassium channel, a member of the voltage activated Kv family, has long intracellular regions. By mutagenesis studies and expression in oocytes, we identify residues in both the N- and C-terminal regions that contribute to determining activation kinetics of this channel. It seems that the C-terminus wraps around the N-terminus and interacts with it functionally. The voltage-activated ether-a-go-go (eag) channels also have long intracellular regions. Despite considerable homology, eag1 and eag2 channels display different activation kinetics. By making chimeras between these channels and again expressing in oocytes, we show that residues in both the N-terminal region and the membrane-spanning region are involved in determining these differences in activation kinetics. The intracellular N- and C-terminal regions are likely to continue to prove fertile regions in future investigations into the functioning of ion channels.Presented at the Biophysical Society Meeting on Ion channels—from structure to disease held in May 2003, Rennes, France     

The problem of nonstationary ion fluxes in excitable membranes

Time-dependent electrodiffusion through a membrane is analysed within a simple model treating the boundary-layers in a consistent manner. It is shown that time-independent reversal potentials for the ion fluxes exist only under steady-state conditions. We argue that this result holds very generally. Therefore nonstationary effects like ion storage and depletion inside the membrane should not contribute to the phenomena of excitability.Glossary of Symbols A _mv [V] functional cf. Equation (3) - C membrane capacitance - d one half the thickness of the membrane - F[V] functional cf. Equation (1) - g _i electrochemical potential inside membrane - g _i electrochemical potentials outside membrane at x ±d, respectively - i (index) refers to i-th ionic species - J electric current across membrane - j = j ^} = j ^< current density measured by external electrodes - j _i (x) current density inside membrane in x-direction - j _i ^inst(x) instantaneous current density - J _i ^stat steady-state current density - k Boltzmann constant - m (index) is used in Sec. 2 to denote the independent diffusion currents - n ^< ionic strength of electrolyte at x = - - n _i density of ions inside membrane - n _i density of ions outside membrane at x = ±, respectively - Q charge per unit area of boundary layers at x ± d, respectively - Q ₀ fixed charge per unit area of membrane - q elementary charge - q _i ionic charges - T temperature - it time - V membrane potential (= (-)-()) - V _i Nernst potential - V potential drops inside boundary layers (can be neglected, see Appendix II) - V ^± potential steps at x = ± d, cf. Equation (29) - V ₀ = V ^–-V ⁺ - w _i activation energy inside membrane - x spatial coordinate perpendicular to membrane - y, z spatial coordinates parallel to membrane - dielecric constant - ₀ dielectric constant of electrolyte solution ( 80) - _m dielectric constant of membrane ( 5) - (x) electrostatic potential - charge density of boundary layers - ⁰ fixed charge density inside membrane - spatial average, cf. Equation (12)     

蜘蛛毒素的研究进展及其相关应用

蜘蛛毒素含有多种化学成分,除毒性作用外这些物质对机体产生多重影响并具有多种活性。近年来,大量新的毒性组分不断被分离纯化,其结构和功能性作用被广泛深入研究,蜘蛛毒素成为了生物毒素领域新的研究热点。本文对蜘蛛毒素在离子通道作用机制方面的最新研究进展进行了阐述,同时还探讨了蜘蛛毒素在医学实践中新的应用和发展。     

Ion channels in plants: From bioelectricity,via signaling,to behavioral actions

In his recent opus magnum review paper published in the October issue of Physiology Reviews, Rainer Hedrich summarized the field of plant ion channels.¹ He started from the earliest electric recordings initiated by Charles Darwin of carnivorous Dionaea muscipula,^1,2 known as Venus flytrap, and covered the topic extensively up to the most recent discoveries on Shaker-type potassium channels, anion channels of SLAC/SLAH families, and ligand-activated channels of glutamate receptor-like type (GLR) and cyclic nucleotide-gated channels (CNGC).¹     

离子通道与人类遗传病

细胞膜离子通道结构和功能正常是细胞进行生理活动的基础,对离子通道功能具有决定性意义的特定位点的突变导致其开放、关闭或激活、失活功能异常,引起组织机能紊乱,形成各种遗传性疾病。本文从水通道蛋白,钙通道,钠通道,钾通道等多种通道蛋白引起的遗传病的现象以及机理做较深入的阐述。     

Statistical properties of ion channel records. Part I: relationship to the macroscopic current

Macroscopic ion channel current can be derived by summation of the stochastic records of individual channel currents. In this paper, we present two probability density functions of single channel records that can uniquely determine the macroscopic current regardless of other statistical properties of records or the stochastic model of channel gating (presented often with stationary Markov models). We show that H(t), probability density function of channel opening events (introduced explicitly in this paper), and D(t), probability density function of the open duration (sometimes has named dwell time distribution as well), determine the normalized macroscopic current, G(t), through G(t) = P(t) - H(t) * Q(t) where P(t) is the cumulative density function of H(t), Q(t) is the cumulative density function of D(t), * is the symbol of convolution integral and G(t) is the macroscopic current divided by the amplitude of single channel current and the number of single channel sweeps. Compared to other equations for the macroscopic current, here the macroscopic current is expressed only in terms of the statistical properties of single channel current and not the stochastic model of ion channel gating or a conditioned form of macroscopic current. Single channel currents of an inactivating BK channel were used to validate this relationship experimentally too. In this paper, we used median filters as they can remove the unwanted noise without smoothing the transitions between open and closed states (compare to low pass filters). This filtering leads to more accurate measurement of transition times and less amount of missed events.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca_V3.1 channel and the high-voltage activating Ca_V1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1–S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1–S4 and S5–S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.Junying Li and Louisa Stevens contributed equally to this work.     

The estimated elastic constants for a single bone osteonal lamella

Micromechanical estimates of the elastic constants for a single bone osteonal lamella and its substructures are reported. These estimates of elastic constants are accomplished at three distinct and organized hierarchical levels, that of a mineralized collagen fibril, a collagen fiber, and a single lamella. The smallest collagen structure is the collagen fibril whose diameter is the order of 20 nm. The next structural level is the collagen fiber with a diameter of the order of 80 nm. A lamella is a laminate structure, composed of multiple collagen fibers with embedded minerals and consists of several laminates. The thickness of one laminate in the lamella is approximately 130 nm. All collagen fibers in a laminate in the lamella are oriented in one direction. However, the laminates rotate relative to the adjacent laminates. In this work, all collagen fibers in a lamella are assumed to be aligned in the longitudinal direction. This kind of bone with all collagen fibers aligned in one direction is called a parallel fibered bone. The effective elastic constants for a parallel fibered bone are estimated by assuming periodic substructures. These results provide a database for estimating the anisotropic poroelastic constants of an osteon and also provide a database for building mathematical or computational models in bone micromechanics, such as bone damage mechanics and bone poroelasticity.     

A Subsequent Fit of Time Series and Amplitude Histogram of Patch-Clamp Records Reveals Rate Constants up to 1 per Microsecond

Fast gating in time series of patch-clamp current demands powerful tools to reveal the rate constants of the adequate Hidden Markov model. Here, two approaches are presented to improve the temporal resolution of the direct fit of the time series. First, the prediction algorithm is extended to include intermediate currents between the nominal levels as caused by the anti-aliasing filter. This approach can reveal rate constants that are about 4 times higher than the corner frequency of the anti-aliasing filter. However, this approach is restricted to time series with very low noise. Second, the direct fit of the time series is combined with a beta fit, i.e., a fit of the deviations of the amplitude histogram from the Gaussian distribution. Since the “theoretical” amplitude histograms for higher-order Bessel filters cannot be calculated by analytical tools, they are generated from simulated time series. In a first approach, a simultaneous fit of the time series and of the Beta fit is tested. This simultaneous fit, however, inherits the drawbacks of both approaches, not the benefits. More successful is a subsequent fit: The fit of the time series yields a set of rate constants. The subsequent Beta fit uses the slow rate constants of the fit of the time series as fixed parameters and the optimization algorithm is restricted to the fast ones. The efficiency of this approach is illustrated by means of time series obtained from simulation and from the dominant K⁺ channel in Chara. This shows that temporal resolution can reach the microsecond range.     

A novel,radiolabel-free pulse chase strategy to study Kir3 channel ontogeny

Abstract

Kir3 channels are essential regulators of cellular excitability, maintaining cells at resting membrane potentials. While much research has been dedicated to elucidating the mechanisms regulating Kir3 channel gating, little is known regarding the channel’s early associations with signaling partners, its stability at the plasma membrane or mechanisms regulating its internalization and degradation. To address these issues we have established an inducible Kir3.1 cell line that allows monitoring of a discrete “pulse” of channel as it progresses along the biosynthetic pathway. Using this system, we have been able to track Kir3 maturation and the influence of partner subunits on Kir3 lifetime and stability. Of note, we show that Kir3.1, in the absence of trafficking partner subunits, can exit the endoplasmic reticulum (ER) and reach the Golgi (though not the plasma membrane), and that expression of Kir3.3 subunits drastically reduced levels of Kir3.1 in the cell. We also show that interfering with trafficking from the ER to Golgi has a pronounced inhibitory effect on Kir3.1-Kir3.2 interactions, suggesting that this complex is stabilized either en route to the Golgi or in the Golgi itself. Finally, we showed that the Kir3 channel can reach the cell surface as early as 6?h post-induction and that removal of cell surface-localized channel occurs within 48?h. This system can be adapted to study the life cycle of any cellular protein without the confounds associated with radioactive labeling or the complications noted with expressing supraphysiological levels of proteins.     

Molecular regions responsible for differences in activation between heag channels

The ether-a-go-go potassium channels heag1 and heag2 are highly homologous; however, the activation properties between the two channels are different. We have studied the molecular regions that determine differences in activation properties by making chimeras between the two channels, expressing them in oocytes, and recording currents with two-electrode voltage-clamp. The activation time course has an initial sigmoidal component dependent on the Cole-Moore shift, followed by a faster component. We show that not only is the extreme N terminus involved in differences between heag1 and heag2 channels, but also the PAS domain itself. Also multiple regions of the membrane-spanning part of the channel appear to be involved, with different regions involved for the early and late time courses, reflecting their different mechanisms. The later time course involved S1 and P-S6 regions. Taken together, our data show that activation involves multiple regions of the N terminal region and membrane-spanning regions of the channel.     

Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

The Boltzmann equation in molecular biology

In the 1870's, Ludwig Boltzmann proposed a simple equation that was based on the notion of atoms and molecules and that defined the probability of finding a molecule in a given state. Several years later, the Boltzmann equation was developed and used to calculate the equilibrium potential of an ion species that is permeant through membrane channels and to describe conformational changes of biological molecules involved in different mechanisms including: open probability of ion channels, effect of molecular crowding on protein conformation, biochemical reactions and cell proliferation. The aim of this review is to trace the history of the developments of the Boltzmann equation that account for the behaviour of proteins involved in molecular biology and physiology.     

The use of non-physiological conditions to isolate fetal cells from maternal blood

Fetal cells are always present in maternal blood starting in the first trimester of pregnancy, however a rapid, simple, and consistent procedure for their isolation for prenatal non-invasive genetic investigation is still lacking. Sensitivity and recovery of fetal cells is jeopardized by the minute amount of circulating fetal cells and their loss during the enrichment procedure. We report here a single-step approach to isolate fetal cells from maternal blood which relies on the use of non-physiological conditions to modify cell densities before their separation in a density gradient and in a newly developed cell separation device. Isolated fetal cells have been investigated using cytochemistry, Soret band absorption microscopy, monoclonal antibodies for epsilon- and gamma-chain-Hb, monoclonal antibody for i-antigen, and by fluorescence in situ hybridization (FISH). Fetal cells were always detected in all 105 maternal blood samples investigated and fetal aneuploidies were correctly diagnosed by FISH, in a pilot study of pathological pregnancies, in fetal cells isolated from maternal blood obtained either before or after invasive procedure.     

Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. II. Model interpretation of ion exchange in lysozyme chromatography

In this paper we present a model for the ion exchange effects in protein adsorption. The model is applied to chromatography of lysozyme on strong cation exchanger ‘mono S’. The experimental and general thermodynamic aspects have been discussed in Part 1, the preceding paper. The main modelling assumptions are (i) the charge regulation is confined to the small layer of contact between adsorbed protein and exchanger surface, (ii) the contact layer as a whole is electroneutral and (iii) the number of protein acid/base groups and exchanger surface acid groups which participate in the ion exchange is proportional to the area of the contact layer. The model is fitted to the experimental data by adjustment of only two or three parameters. The experimental co-adsorption numbers are very well reproduced. A few conspicuous features emerge: (i) the number of protein acid/base groups and exchanger surface acid groups in the contact layer varies with the medium conditions, such that the number is higher when the interaction between protein and exchanger surface is stronger. (ii) There is indirect evidence for structural alterations in the upper layers of the exchanger surface: the adsorbed protein is probably partly ‘buried’ in the surface.     

Extracellular proton sensing of the rat gustatory cyclic nucleotide-gated channel

Elevations of the intracellular levels of cyclic nucleotides appear to cause the cation influx through gustatory cyclic nucleotide-gated (CNGgust) channels expressed in taste cells. Although changes in the oral pH may directly regulate the activity of the CNGgust channel, the mechanism of pH-dependent control of the channel is not understood. In the present study, we combined the whole-cell patch-clamp recording and the site-directed mutagenesis to investigate the effect of extracellular pH on the ion permeation through CNGgust channels expressed in HEK293 cells. Extracellular acidification strongly inhibited ion permeation through open CNGgust channels. Mutation of Glu(289) remarkably attenuated the pH-dependence of the channel, suggesting that Glu(289) in the pore-forming region is a major proton acceptor site. However, the mutant E289A-CNGgust channel possesses the other residual protonation/deprotonation site. The channel activity, tightly regulated by pH(o) and [cNMP](i), suggests the involvement of its pH(o)-dependent ion permeation in taste signal transduction events.