Gating properties of channels formed by colicin Ia in planar lipid bilayer membranes

Summary Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (>+10 mV) are applied to thecis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant () of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric aroundV _o (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction inn, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be itcis orrans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change inn. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open,F _(v). For a given voltage,F _(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino-acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.     

Transients in toad skin: Short circuit current and ionic fluxes related to inner sodium substitution by monovalent cations

Summary When the Na electrochemical potential difference across the skin (_Na) is altered by perturbing the transmembrane electrical potential difference or the external Na concentration, effects on transport and associated oxygen consumption can be described by the formalism of linear nonequilibrium thermodynamics (Vieira, Caplan & Essig, 1972,J. Gen. Physiol. 59:77; Danisi & Lacaz-Vieira, 1974,J. Gen. Physiol. 64:372; Procópio and Lacaz-Vieira, 1977,J. Membrane Biol. 35:219). We now show that with modifications of _Na by substitution of Li or choline for Na in the inner bathing solution, this formalism is no longer applicable. Inner Na by K substitution ((Na×K) _i ) causes profound alterations in short-circuit current (SCC),J _Na ⁱⁿ , K efflux (J _K ^eff ) and PD. SCC drops transiently after (Na×K) _i in Cl and in SO₄ media, increasing subsequently. In Cl medium, following the initial transient, there is a late decline in SCC toward a steady state. The rate of SCC decline in Cl medium is more pronounced than that observed in SO₄ medium. (Na×K) _i causes a transient increase inJ _Na ⁱⁿ with a peak synchronous to the minimum in SCC, both in Cl and in SO₄ media. This was interpreted as due to depolarization of the inner membrane. In SO₄ medium, following the peak observed after (Na×K) _i J _Na ⁱⁿ drops, to increase again toward a steady state in which SCC andJ _Na ⁱⁿ are not statistically different, resembling the control condition before (Na×K) _i . In Cl medium, however, theJ _Na ⁱⁿ steady state is approximately 100% higher than SCC. This difference is due to an important K efflux (J _K ^eff ), which builds up progressively after the substitution. The apparent K permeability [J _K ^eff /(K _i )] is of comparable magnitude in Cl and in SO₄ media before (Na×K) _i , the apparent K permeability increases one order of magnitude as compared to the control condition before the ionic substitution. In Cl medium, the high levels ofJ _Na ⁱⁿ and ofJ _K ^eff observed in the steady state after (Na×K) _i were interpreted as being a consequence of cell swelling. SCC and PD follow very different temporal patterns after (Na×K) _i which are characterized by transients in SCC and a simple fall in PD. Reasons for these differences are discussed.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone,Stoichactis helianthus

Summary When present at nanomolar concentrations on one side of a lipid bilayer membrane,helianthus toxin (a protein of mol wt16,000) increases enormously membrane permeability to ions and nonelectrolytes by forming channels in the membrane. Membranes containing sphingomyelin are especially sensitive to toxin, but sphingomyelin isnot required for toxin action. Conductance is proportional to about the 4th power of toxin concentration. Single channel conductances are approximately 2×10^–10 mho in 0.1m KCl. Toxin-treated membranes are more permeable to K⁺ and Na⁺ than to Cl^– and SO ₄ ⁼ , but the degree of selectivity is pH dependent. Above pH 7 membranes are almost ideally selective for K⁺ with respect to SO ₄ ⁼ , whereas below pH 4 they are poorly selective. The channels show classical molecular sieving for urea, glycerol, glucose, and sucrose — implying a channel radius >5 Å. In symmetrical salt solutions above pH 7, theI–V characteristic of the channel shows significant rectification: below pH 5 there is very little rectfication. Because of the effects of pH on ion selectivity and channel conductance, and also because of the rectification in symmetrical salt solutions and the effect of pH on this, we conclude that there are titratable negative charge groups in the channel modulating ion permeability and selectivity. Since pH changes on the side containing the toxin are effective whereas pH changes on the opposite side are not, we place these negative charges near the mouth of the channel facing the solution to which toxin was added.     

Memory in Ion Channel Kinetics

Acta Biotheoretica - Ion channels are transport proteins present in the lipid bilayers of biological membranes. They are involved in many physiological processes, such as the generation of nerve...     

The Resting Potential of Mouse Leydig Cells: Role of an Electrogenic Na+/K+ Pump

Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 +/- 1.2 mV (mean +/- SEM, n = 159), at 25 degrees C, and an input resistance larger than 2.7 x 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 +/- 3.2 mV (n = 9) and 23.1 +/- 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 +/- 0.4 pA (n = 21) at 25 degrees C. Vm also varied strongly with temperature, reaching values as low as -9.2 +/- 1.2 mV (n = 22) at 15 degrees C. Taking the pump current at 25 degrees C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 +/- 1.2 mV (n = 159) observed.     

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X₁–P2X₇) and seven heteromeric receptors (P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, P2X_4/6, P2X_4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca²⁺]_i and testosterone secretion, supporting the hypothesis that Ca²⁺ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X₂. In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X₂, P2X₄, P2X₆, and P2X₇ subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2 ± 5.9%) and 3 μM ivermectin a decrease (64.2 ± 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X₄ subunits. P2X₇ receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X_2/4/6, is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X₂ subunit.     

A Fluorescence‐Based Sensor Assay that Monitors General Protein Aggregation in Human Cells

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ‐peptide. Exposure to proteome destabilizers lead to re‐localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide‐genetic screens to identify genes and pathways involved in protein misfolding and aggregation.     

Effect of gramine on the feeding behavior of the aphids Schizaphis graminum and Rhopalosiphum padi

The effects of the indole alkaloid gramine on the behavior of the aphids Schizaphis graminum and Rhopalosiphum padi feeding on barley seedlings and on artificial diets were studied. On barley cv. F. Union, which lacks gramine, S. graminum ingested from phloem tissue for 30–80 min and non-phloem for 20–40 min, over a period of 3 h. In cultivar Datil S, gramine was found only in the epidermis and parenchyma mesophyll cells. On this cultivar, the non-phloem wave form of S. graminum was not observed. On F. Union, R. padi ingested non-phloem with short periods of ingestion from phloem. On cv. Datil S, this aphid did not show the non-phloem wave form. In experiments performed with cv. F. Union seedlings that contained exogenous gramine S. graminum did not ingest from phloem. Exogenous gramine was found only in the vascular bundles. It is suggested that gramine content and location may affect the feeding behavior of aphids in barley.

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Résumé Les effects de la gramine sur le comportement alimentaire des pucerons Schizaphis graminum et Rhopalosiphum padi on été étudiés en plantules d'orge (Hordeum distichum) et aussi avec des diètes artificielles à l'aide d'un moniteur électronique. S.graminum se nourri pendant 30–80 minutes à partir du phloème et il a pris 20–40 minutes en se nourrisant d'autres tissues divers, d'un total de 3 heures d'exposition à la plante. Dans la culture Datil S la gramine a été trouvée seulement dans les cellules de l'epidermis et du parenchyme du mesophyle. Dans ce cas, l'onde non-phloematique de S. graminum, n'a été pas observé; le temps pour le première onde X a été plus prolongé par rapport à la culture F. Union. Dans la culture F. Union, R. padi se nourri principalement de tissue non-phloematique (35–100 minutes) et du phloème par des brèves périodes (15–25 minutes). Dans le cas de la culture Datil S ce puceron n'a pas montré l'onde X. D'autre part, quand les plantules de F. Union ont été poussées dans des solution avec gramine, S. graminum ne se nourri pas du phloeme, tandis que R. padi montre un comportement similaire a celui-ce observé avec plantules sans gramine. La gramine absorbé par les plantules a été retrouvée seulement dans les faisceaux vasculaires. D'autre part, la gramine dans des diètes artificielles agit comme repulsif alimentaire à partir de concentrations de 0,5 mM. Avec des diètes contenant 3 mM de gramine l'alimentation des pucerons a été inhibée. Pourtant, ces resultats sugèrent que le contenuet et la localisation de la gramine peuvent modifier le comportement alimentaire des pucerons en plantules d'orge.