Cation channels in the plasma membrane of retinal rod outer segments activated by cGMP

Properties of cGMP-activated cation channels were investigated on isolated patches of the ROS plasma membrane using the "patch clamp" technique. The channels were shown to be characterized by ideal cation selectivity under physiological conditions and are nearly equally permeable for cations of alkaline metals. At the same time they are permeable for some bivalence cations (PNa approximately PCa). Other channel properties are described and their comparative analysis is given. It suggests that cGMP-activated cation channels represent a new type of cation channels.     

Transport of the substance through thin membranes with narrow pores after the preceding stage of surface diffusion

A model of the substance transport through thin membranes is discussed. Existence of a step of the surface diffusion of permeable particles is assumed preceding its entrance into membrane channel. An expression is given for the stationary flow, which has earlier been obtained for other transport models. It is shown that the permeant affinity to the membrane surface is the main condition for the validity of the scheme in question.     

Osmotic Flow of Water across Permeable Cellulose Membranes

Direct measurements have been made of the net volume flow through cellulose membranes, due to a difference in concentration of solute across the membrane. The aqueous solutions used included solutes ranging in size from deuterated water to bovine serum albumin. For the semipermeable membrane (impermeable to the solute) the volume flow produced by the osmotic gradient is equal to the flow produced by the hydrostatic pressure RT ΔC, as given by the van't Hoff relationship. In the case in which the membrane is permeable to the solute, the net volume flow is reduced, as predicted by the theory of Staverman, based on the thermodynamics of the steady state. A means of establishing the amount of this reduction is given, depending on the size of the solute molecule and the effective pore radius of the membrane. With the help of these results, a hypothetical biological membrane moving water by osmotic and hydrostatic pressure gradients is discussed.     

Landscape genetic connectivity in European wildcat (Felis silvestris silvestris): a matter of food,shelters and demographic status of populations

Understanding landscape impacts on gene flow is necessary to plan comprehensive management and conservation strategies of both the species of interest and its habitat. Nevertheless, only a few studies have focused on the landscape genetic connectivity of the European wildcat, an umbrella species whose conservation allows the preservation of numerous other species and habitat types. We applied population and landscape genetics approaches, using genotypes at 30 microsatellites from 232 genetically-identified wildcats to determine if, and how, landscape impacted gene flow throughout France. Analyses were performed independently within two population patches: the historical north-eastern patch and the central patch considered as the colonization front. Our results showed that gene flow occurred at large spatial scales but also revealed significant spatial genetic structures within population patches. In both population patches, arable areas, pastures and permanent grasslands and lowly fragmented forested areas were permeable to gene flow, suggesting that shelters and dietary resources are among the most important parameters for French wildcat landscape connectivity, while distance to forest had no detectable effect. Anthropized areas appeared highly resistant in the north-eastern patch but highly permeable in the central patch, suggesting that different behaviours can be observed according to the demographic context in which populations are found. In line with this hypothesis, spatial distribution of genetic variability seemed uneven in the north-eastern patch and more clinal in the central patch. Overall, our results highlighted that European wildcat might be a habitat generalist species and also the importance of performing spatial replication in landscape genetics studies.

     

The role of membrane patch size and flow in regulating a proteolytic feedback threshold on a membrane: possible application in blood coagulation

Positive feedback controls in proteolytic systems are characterized by thresholds which are regulated by the concentration of the initial stimulus and the kinetic parameters for enzyme generation and inhibition. Significant complexity is added when a positive feedback is localized on a membrane in contact with a flowing medium, such as seen in the early steps of blood coagulation. A partial differential equation model of an archetypal feedback loop is examined in which a proteolytic enzyme catalyzes its own formation from a zymogen on a membrane in contact with a flowing medium. As predicted from prior solution-phase and membrane-phase analyses, the threshold conditions for activation of the system are regulated by the kinetics of enzyme generation and inhibition and by the density of reactant-binding sites on the membrane; but the present analysis also establishes how the feedback threshold is controlled by the flow rate of the adjacent medium and the physical size of the membrane patch on which the feedback loop is localized. For given systems of particular kinetic properties, lower flow rates or larger active patches of membrane can result in the activation threshold being exceeded, whereas higher flow rates or smaller membrane patches can prevent initiation. In addition to numerical simulation, a simplified non-flowing model is analyzed to formulate an approximate mathematical statement of the dependence of the minimum activatable patch size on the kinetic and other parameters.     

The osmotic flow of an electrolyte through a charged porous membrane

A pore model in which the pore wall has a continuous distribution of electrical charge is used to investigate the osmotic flow through a charged permeable membrane separating electrolyte solutions of unequal concentrations. The pore is treated as a long, circular, cylindrical duct. The analysis is based on a continuum formulation in which a dilute electrolyte solution is described by the coupled Nernst-Planck/Poisson creeping flow equations. Account is taken of the significant size of the electrolyte ions (assumed to be rigid spheres) when compared with the diameter of the membrane pores. Analytical solutions for the ion concentrations, hydrostatic pressure and electrostatic potential in the electrolyte solutions are given and an intra-pore flow solution is derived. A mathematical expression for the osmotic reflection coefficient as a function of the solute ion: pore diameter ratio λ and the solute fluxes is obtained. Approximate solutions are quoted which relate the solute fluxes and the solution electrostatic potentials at the membrane surfaces to the bulk solution concentrations, the membrane pore charge and pore geometry. The osmotic reflection coefficient is thus determined as a function of these parameters.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

cGMP对原代培养猪冠状动脉平滑肌细胞钙激活钾通道的作用

３′,５′－环－磷酸鸟苷（ｃＧＭＰ）具有激活血管平滑肌细胞膜上钙激活钾通道（ＫＣａ通道）的作用,从而引起血管平滑肌细胞的舒张。但ｃＧＭＰ激活ＫＣａ物机制存在争论。本工作应用膜片箝技术以原代培养猪冠状动脉平滑肌细胞为对象研究了ｃＧＭＰ影响ＫＣａ通道的机制。实验结果显示：（１）在ｃｅｌｌ－ａｔｔａｃｈｅｄ膜片方式下,当溶液内游离Ｃａ＾２＋浓度为１０＾－７ｍｏｌ／Ｌ,膜电位为＋７０ｍＶ时,不同浓度的ｃＧ     

Ionic concentration profiles and charge distribution for the domain of a polarized spherical cell

Solutions of the Poisson-Boltzmann equation yield potential profiles and equilibrium distributions of ions on either side of a spherical shell membrane across which there exists a separation of ionic charges. For the special case in which the membrane is permeable to only one ion the total charge separation is analyzed in terms of the potential difference given by the Nernst equation. Potential profiles and ionic charge distributions are also given for situations involving a uniform distribution of fixed charges within the membrane.     

Voltage-dependent Ba2+ permeation through store-operated CRAC channels: implications for channel selectivity

Store-operated Ca2+ entry through CRAC channels is a major route for Ca2+ influx in non-excitable cells. Studies on store-operated channel selectivity using fluorescent dyes have found that the channels are impermeable to Ba2+. Furthermore, in such studies, agonists have been reported to increase Ba2+ influx, leading to the conclusion that additional Ca2+ entry pathways (permeable to Ba2+) co-exist with the Ba2+-impermeable store-operated channels. However, patch clamp experiments demonstrate that CRAC channels are permeable to Ba2+. We have addressed this paradox using fluorescence measurements and whole cell patch clamp recordings of ICRAC. In store-depleted cells loaded with fura 2, Ba2+ application results in a slower and smaller rise in fluorescence than is the case with Ca2+. Ba2+, unlike Ca2+, depolarises the membrane potential by approximately 40 mV, due to rapid block of an inwardly rectifying K+ current. Although Ba2+ permeates CRAC channels at very negative potentials in patch clamp recordings, Ba2+ permeation is steeply voltage-dependent. This combination of Ba2+-dependent depolarisation and voltage-dependent Ba2+ permeation accounts for the apparent lack of Ba2+ permeation through store-operated channels seen in fluorescence experiments. Our findings identify major limitations with the use of Ba2+ as a surrogate for Ca2+ in probing Ca2+ entry pathways and raise the possibility that some of the previous reports proposing multiple Ca2+ entry pathways based on Ba2+ entry into fura 2-loaded cells could be explained by voltage-dependent Ba2+ permeation through CRAC channels.     

Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposome could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential, Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.     

Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposomes could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential. Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.     

Comparison of discrete models of charge transfer in thin membranes. I. Stationary regime]

The model of the ion transport through thin membranes is discussed. It is assumed that there exist some steps of the ion transfer in the membrane volume. Theory of absolute rate of processes is used to derive an equation for stationary transmembrane flow. As a result the thory parameters are connected with the form of potential energy curve of permeable ions into the membrane phase.     

Barriers to water flow in vasopressin-treated toad urinary bladder

Summary Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.     

Nonselective ionic channels inAplysia neurones

Summary Single-channel recordings from outside-out patches ofAplysia neurones in K-free solutions revealed the presence in most membrane patches of ionic channels showing surprising selectivity properties, as deduced from reversal potential measurements. After complete substitution of external NaCl by mannitol (in the presence of internal CsCl), these channels are more permeable to Cl than to Cs, but are also slightly permeable to Cs:P _Cl/P _Cs=4. Furthermore, in the presence of external NaCl, their ability to discriminate cations from anions seems lower than in external mannitol. Substitutions of external Cl by various anions showed that the channels are more permeable to NO₃ than to Cl, and that they are appreciably permeable to isethionate, SO₄ and methanesulfonate. Their elementary conductance is about 100 pS in 600mm symmetrical Cl. However, different conductance states (usually 2 or 3) can often be detected in the same membrane patch. By using voltage ramps, we established theI–V curves corresponding to each of these states and found small but significant differences between the reversal potentials of each state.     

Experimental study of osmosis through a collodion membrane

Experiments were carried out on a collodion membrane in order to study the factors that determine direction and magnitude of net flow of water across a membrane permeable to the solvent and to some of the solutes present. The solutes used were all non-ionic. When only one solute was present and there was no difference of hydrostatic pressure across the membrane, water flowed toward the side where its vapor pressure was lower, but the rate of transfer depended upon the nature of the solute: for a given difference in osmolality across the membrane, the rate increased with the molecular volume of the solute and reached its maximum with the solute to which the membrane was impermeable. These results led to the experimental demonstration that in the presence of two or more solutes of different molecular volumes, of which one at least can diffuse through the barrier, the net transfer of water can take place against its vapor pressure gradient. Some of the physicochemical and physiological implications of the data are discussed.     

Single channel K+ currents from HeLa cells

The extracellular patch-clamp technique was used in order to investigate the presence of ionic channels in HeLa cells, a well-known cultured cell type obtained from an epidermoid carcinoma of the cervix. Under Gigohm-seal conditions, discrete current jumps could be observed with patch electrodes containing KCl. These channels were found to be mainly permeable to K+ and showed multiple levels of conductance. From single-channel I-V curve measurements, a strong rectification effect, characterized by a large inward and no detectable outward current, was observed. For negative membrane potentials (0 to -90 mV), the measured current-voltage relationship was found to be mostly linear, corresponding to a single-channel conductance of 40 pS. An analysis of some selected time records has revealed in addition that the probability of the channel to be in the open state was a function of the KCl concentration in the patch pipette.     

Numerical simulation of the effect of permeability on the hydrodynamics in a parallel-plate coculture flow chamber

To study the effect of the porous membrane permeability on the hydrodynamics in a parallel-plate coculture flow chamber (PPcFC), we demonstrated the permeability of the porous membrane as a function of some parameters, such as porosity, membrane thickness, pore size and shape of the membrane. The effect of permeability on the flow in the PPcFC was analysed using the commercial software – Fluent. Results showed that the permeability was directly proportional to the thickness, the porosity and the pore size of the membrane, and inversely proportional to the surface shape factor. To ensure the best flow pattern, the inlet velocity range was limited by the membrane permeability and fluid viscosity, and then restricted the available magnitudes of shear rate on the permeable membrane. Our findings are helpful in designing and preparing the biomaterials that have adequate mechanical properties for the functional vascular grafts production, and in using of the flow chamber in various investigations.     

On the volume-flow mechanism of phloem transport

Summary A steady-state model of solution flow in a tubular semipermeable membrane is developed for an arbitrary distribution of solute sources and sinks along the translocation path. It is demonstrated that the volume-flow mechanism of phloem transport depends only on the two assumptions: 1. that the plasmalemma of the sieve tube is a differentially permeable membrane, and 2. that sugars are actively secreted into and absorbed from the lumen of the sieve tube. It is shown that in the absence of a pressure gradient, there is a negligible concentration gradient over most of the translocation path. However, in the presence of a pressure gradient a small concentration gradient develops as a result of the continually changing chemical potential of water along the direction of solution flow. For Poiseuille flow the concentration gradient is approximately proportional to the mean stream velocity.     

ADP effects on bleomycin-induced DNA repair synthesis and adenylate kinase activity in permeable mouse sarcoma cells

DNA repair in bleomycin-pretreated, permeable mouse sarcoma (SR-C3H/He) cells requires ATP for at least two steps, the repair DNA synthesis step and the repair patch ligation step. ADP can apparently replace ATP in both steps. Maximal, 1.5-2 fold stimulation of repair DNA synthesis was observed with 5-10 mM ADP as well as 2.5-5 mM ATP. Repair patch ligation in the presence of 2.5 mM ADP occurred at almost the same high efficiency as it did in the presence of ATP. The ADP effect on DNA repair patch ligation was attributed to ATP formed from ADP by adenylate kinase in permeable cells, however the ADP effect on repair DNA synthesis could not be attributed solely to the formation of ATP in the same manner.