Steady-state diffusion and the cell resting potential

The steady-state diffusion of ions through separate, selective channels is described according to irreversible thermodynamics. Ion fluxes thus obtained are the same as those in the parallel conductance model. The equivalent electric circuit set up to describe the system has its electromotive forces expressed by the chemical potentials of the diffusing ions. The expression obtained for the potential differs from the Goldman-Hodgkin-Katz formula, and is reputed to be more accurate. In order for the passive diffusion flows to remain steady, active transport mechanisms must pump the ions up their electrochemical potentials. Such pumps have been incorporated into the equivalent circuit. They supply energy lost in the dissipation caused by preexisting passive forces without affecting the potential, which can thus hardly be called passive diffusion potential. Ion pumps can also create an electric potential in excess of that by passive forces, especially when secondary active transport is involved. The same equivalent circuit is, however, able to describe the whole range of seemingly different situations – from passive diffusion of an electrolyte to active extrusion of anions from the living cell. It has been applied to explain the measured plasma membrane potential of cells, especially those whose potential does not behave as the potassium electrode. Received: 6 July 1998 / Revised version: 7 November 1998 / Accepted: 10 November 1998     

豚鼠耳蜗螺旋动脉平滑肌细胞和内皮细胞静息膜电位特性

目的:多种内耳疾病和内耳微循环障碍有关,但目前对提供内耳主要血供的耳蜗螺旋动脉平滑肌(SMC)和内皮细胞(EC)的生理学特性还不十分清楚,需要进一步研究。方法:本研究采用双细胞内微电极记录技术和细胞荧光染色技术,研究耳蜗螺旋动脉平滑肌和内皮细胞的膜电位特性和细胞间的通讯联系。结果:研究发现耳蜗螺旋动脉SMC和EC具有高、低两种静息膜电位(RP)状态,两种静息膜电位状态的细胞对乙酰胆碱和高K+的反应完全不同。双微电极可同时记录到EC-ECS、MC-SMC和SMC-EC不同类型的细胞,两个细胞的静息膜电位也可以是双高RP、双低RP和一高一低RP。实验所记录的一高一低RP均是SMC-EC类型,而且EC初始膜电位均为高电位,SMC初始膜电位均为低电位。而双高RP和双低RP可以是SMC-SMC或EC-EC或SMC-EC类型。结论:结果表明耳蜗螺旋动脉的SMC和EC在0.3~0.5 mm的范围内,同类细胞之间有很好的通讯联系,能很好的保持功能的协同和一致,血管壁异类细胞则不同。     

Ion flow through biomembranes

Numerous biomembranes exhibit a sensitivity to changes in electrical potential greater than predicted as possible from the classical application of the Boltzmann relation, a phenomenon which has long defied explanation, the actual sensitivity of some Na⁺ channels being many times greater than the classical limit. This paper explains, using a minimum of mathematics, how the very rapid gating effect of adsorbed Ca²⁺ (or other impermeable divalent cations) can directly affect the conductance of channels, and thus interact with the electric field within the channel to produce a change in the potential across the channel’s gate much greater than the change in the membrane potential, with a corresponding change in the fraction of open conformational gates and change in conductance. These results are not in conflict with the Boltzmann relation, the necessary energy being made available from the total potential difference across the membrane by a long unrecognized stochastic process; the full mathematical theory is given in cited references.     

Ion permeation of pores in model membranes: selectivity,fluctuations and the role of surface charge

Fluctuation of surface charge on pore walls provides a realistic, additional mechanism for generating fluctuation of ionic current and ionic selectivity in narrow pores.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

Contribution of a H+ pump in determining the resting potential of neuroblastoma cells

The aim of this work was to examine the effects of changes in external K⁺ concentration (K _o ) around its physiological value, of various K⁺ channels blockers, including internal Cs⁺, of vacuolar H⁺-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K⁺ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (K _o =5 mm) the membrane potential was –60±1 mV. It was unchanged when K _o was decreased to 1 mm and was depolarized by 4±1 mV when K_o was increased to 10 mm. (ii) Internal Cs⁺ depolarized the membrane by 21±3 mV. (iii) The internal application of the vacuolar H⁺-ATPase inhibitors N-ethylmaleimide (NEM), NO ₃ ^– and bafilomycin A1 (BFA) depolarized the membrane by 15±2, 18±2 and 16±2 mV, respectively, (iv) When NEM or BFA were added to the internal medium containing Cs⁺, the membrane was depolarized by 45±1 and 42±2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K⁺ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K⁺ channels and an electrogenic vacuolar type H⁺-ATPase.This work was supported by a grant from INSERM (CRE 91 0906).     

豚鼠冠状动脉微动脉细胞静息膜电位的分布特性及机制

目的:观察冠状动脉微动脉细胞静息膜电位(RP)的分布特性及形成机制.方法:离体豚鼠冠状动脉微动脉(直径小于100 μm)上,应用细胞内微电极技术记录细胞RP.结果:①成功记录到112个细胞,细胞平均RP为(-65±4.2)mV,应用高斯函数拟合后细胞RP呈双峰状分布,两个峰值分别为-43和-74 mV,分别称为高和低R...     

Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells

The membrane electric effects of N,N'-dicyclohexyl-carbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (ARexp) of [3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20-30 mV hyperpolarization of resting potential (delta psi p) of MELC, which is abolished when the electrochemical equilibrium potential of K+ (EK) is brought close to zero. DCCD and vanadate turned out to have distinct targets on the plasma membrane of MELC (an H+ pump and the Na+,K(+)-ATPase, respectively). Measurements of ARexp showed that: (i) patch-clamp measurements of delta psi p were equivalent to those based on ARexp of antimycin-pretreated cells (ARANT); (ii) DCCD produced a strong increase in ARANT, that was antagonized by carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and diethylstilbestrol (DES); (iii) vanadate determined a marked increase in ARANT that was insensitive to FCCP, but antagonized by ouabain; (iv) incubation in high K+ medium (HK) brought ARANT to 1.0 in the controls, but did not lower this ratio below 3.0 in the presence of DCCD or vanadate; (v) the total amount of TPP+ taken up by the cells was in any case water extractable by a freezing and thawing procedure. On the whole, our data indicate that DCCD and vanadate hyperpolarize the MELC by increasing the K+ conductance and, at the same time, enhance the TPP+ binding, probably by changing the electrostatic potential profile of the plasma membrane. These effects seem to involve functional modifications of the target pumps, apparently related to the ion-occluding state of these enzymes.     

Role of diffusion potential on the flow of angiotensin II,bradykinin and related molecules through cellophane membranes

The diffusion of electrically charged peptides (angiotensin II, bradykinin and [Suc¹]angiotensin II) across tight cellophane membranes, obtained by different degrees of acetylation, shows a kinetic behaviour which was interpreted in the literature as indicative of the existence of different molecular conformations presenting slow interconversion velocities and different permeabilities across the membrane. A diffusion potential (Δψ) was found to be present across the membrane along diffusion experiments performed in low ionic strength. Upon annihilation of δψ by chemical voltage clamping (by equally increasing the ionic strength on both bathing solutions) the diffusion rate was decreased and the flow followed first order kinetics, indicating a major role of Δψ in the process. As the ionic strength increase could also affect molecular conformation, the role of Δψ on the diffusion of those molecules was tested by fitting flux and Δψ experimental results by an integrated form of Nernst-Planck flux equation. It is concluded that the deviation from first order diffusion kinetics, observed in low ionic strength, is solely due to the diffusion potential, and not to the existence of more than one molecular conformation in aqueous solution. This study was extended to amino acids and other related charged molecules.     

Resting potential of the mouse neuroblastoma cells: II. Significant contribution of the surface potential to the resting potential of the cells under physiological conditions

In the previous paper, we showed that the K⁺ channels of the mouse neuroblastoma cell (clone N-18) are closed at low concentration of external K⁺ ([K⁺]₀) including the physiological concentration for the cells. In the present study, the origin of the resting membrane potential of N-18 cells has been examined. (1) The resting membrane potential of N-18 cells was depolarized by increasing concentration of the polyvalent cations (La³⁺, Fe³⁺, Co²⁺, Ca²⁺, Sr²⁺, Mg²⁺) and by decreasing the pH of the medium. The input membrane resistance was slightly increased during the depolarization. The depolarization was not explained in terms of the diffusion of the cations across the membrane, since the trivalent cations of greater ionic size were effective at much lower concentrations than the divalent cations. The results obtained from the measurements of ⁸⁶Rb efflux suggested that the depolarization cannot be explained in terms of blocking of the K⁺ channels by the cations. (2) An increase in Ca²⁺ concentration from 0.3 to 1.8 mM induced depolarization of about 10 mV at low [K⁺]₀ where the K⁺ channels are closed, but did not induce any depolarization at high [K⁺]₀ where the channels are open. (3) In order to estimate the changes in the zeta-potential, the electrophoretic mobility of N-18 cells was measured under various conditions. There was a close correlation between the changes in the zeta-potential and those in the membrane potential in response to the polyvalent cations and proton. On the other hand, an increase in K⁺-concentration in the medium, which induced a large depolarization in the cells, did not affect the zeta-potential. (4) The results obtained were explained by an electrical circuit model for the membranes of N-18 cells. In this model, an electrical circuit for the membrane part carrying no selective ionic channels, in which changes in the surface potential directly affect the transmembrane potential, is connected in parallel to the usual circuit model representing selective ionic channel systems. It was concluded that the surface potential contributes significantly to the resting membrane potential of N-18 cells at low [K⁺]₀ where the K⁺ channels are closed.     

The relation between the action potential duration,the increase in resting tension,and ATP content during metabolic inhibition in guinea pig ventricular muscles

To investigate whether the action potential duration (APD) or resting tension was dependent on global ATP content, and whether they were preferentially dependent on glycolytic ATP, APD and resting tension were measured under various metabolic inhibition with corresponding measurement of ATP content in guinea pig ventricular muscles. Oxidative phosphorylation was inhibited by either hypoxic perfusion, the perfusion of sodium cyanide, or 2,4-dinitrophenol. Glycolysis was blocked by the perfusion of iodoacetic acid, and hypoxia with variable glycolytic activities was achieved by hypoxic perfusion in the presence of glucose (5, 10, and 50 mM). APD began to decrease when ATP content decreased to less than 3 mM/kg w.w. from the control level of 4.35 mM/kg w.w. APD shortened significantly and resting tension increased steeply, when ATP content decreased below 1 mM/kg w.w. The dependence of APD and the increase in resting tension on ATP content was not affected by the mode of metabolic block, that is, the inhibition of glycolysis and/or oxidative phosphorylation. Though other factors can affect APD and resting tension, we found no evidence of functional ATP compartmentation, with respect to APD and the increase in resting tension during metabolic inhibition.     

静息培养条件下赤霉菌菌丝产素能力的研究

针对赤霉菌生长与产次级代谢产物赤霉素的关系进行摇瓶静息培养研究。在不同菌龄及不同底物浓度的条件下,菌丝产素能力有较大不同。实验数据表明：在菌龄96h,葡萄糖浓度为5gfL时,菌丝静息培养72h后,培养液中赤霉素含量最高,达1100μg／mg;同时单位时间单位菌丝产率也最大,为7．1μg/mg·h;在该菌龄条件下,当葡萄糖浓度为1dL时,赤霉素转化效率最高,为0．8484。该研究有助于进一步优化发酵工艺,降低赤霉素生产成本。     

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.     

Action of tannin on cellular membranes: Novel insights from concerted studies on lipid bilayers and native cells

Hydrolyzable tannin (3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose) has a dual effect on the cell membrane: (1) it binds to a plasmalemmal protein of the Chara corallina cell (C₅₀ = 2.7 ± 0.3 μM) and (2) it forms ionic channels in the lipid membrane. Based on these facts, a molecular model for the interaction of tannins with the cell membrane is proposed. The model suggests that the molecules of hydrolyzable tannin bind electrostatically to the outer groups of the membrane protein responsible for the Ca²⁺-dependent chloride current and blocks it. Some tannin molecules penetrate into the hydrophobic region of the membrane, and when a particular concentration is reached, they form ion-conducting structures selective toward Cl^?.     

The membrane potential of characean cells exposed to amplitude-modulated, low-power 147-MHz radiation

The membrane potential of isolated cells of Chara braunii or Nitella flexilis was monitored while the cells were exposed, at nominal power densities from 2 to 1,000 W/m2, to 147-MHz radiation amplitude modulated at frequencies from 4 to 64 Hz. Phase-sensitive detection was used to seek radiation-correlated changes in the membrane potential, and none were apparent under any of the conditions used in this investigation.     

Tonic activity of parasympathetic efferent nerve fibers hyperpolarizes the resting membrane potential of frog taste cells

We investigated the relationship between the membrane potential of frog taste cells in the fungiform papillae and the tonic discharge of parasympathetic efferent fibers in the glossopharyngeal (GP) nerve. When the parasympathetic preganglionic fibers in the GP nerve were kept intact, the mean membrane potential of Ringer-adapted taste cells was -40 mV but decreased to -31 mV after transecting the preganglionic fibers in the GP nerve and crushing the postganglionic fibers in the papillary nerve. The same result occurred after blocking the nicotinic acetylcholine receptors on parasympathetic ganglion cells in the tongue and blocking the substance P neurokinin-1 (NK-1) receptors in the gustatory efferent synapses. This indicates that the parasympathetic nerve (PSN) hyperpolarizes the membrane potential of frog taste cells by -9 mV. Repetitive stimulation of a transected GP nerve revealed that a -9-mV hyperpolarization of taste cells maintained under the intact GP nerve derives from an approximately 10-Hz discharge of the PSN efferent fibers. The mean frequency of tonic discharges extracellularly recorded from PSN efferent fibers of the taste disks was 9.1 impulses/s. We conclude that the resting membrane potential of frog taste cells is continuously hyperpolarized by on average -9 mV by an approximately 10-Hz tonic discharge from the parasympathetic preganglionic neurons in the medulla oblongata.     

Water movement through Quercus rubra I. Leaf water potential and conductance during polycyclic growth

Two experiments examined simultaneous changes in leaf area (A_L), root length (L_r), stomatal conductance (g_s), leaf water potential (Ψ_L), transpiration and hydraulic plant conductance per unit leaf area (G) during the first three shoot cycles of northern red oak (Quercus rubra L.) grown under favourable and controlled conditions. Each shoot cycle consisted of bud swell, stem elongation, leaf expansion and rest; roots grew almost continuously. The g_s of all leaves decreased substantially while leaves of the newest flush were expanding and increased modestly when seedling leaf area remained constant. Overall, g_s decreased. The Ψ_L of mature leaves decreased during leaf expansion and increased by an equivalent amount during intervening periods. Possible explanations for the paired changes in g_s and Ψ_L are considered. Changes in G closely paralleled those of canopy g_s. These parallel changes during polycyclic seedling growth should act to keep seedling Ψ_L relatively constant as plant size increases and thereby help prevent Ψ_L from dropping to levels that would cause runaway embolism.     

Ephaptic coupling of cardiac cells through the junctional electric potential

Cardiac cells are electrically coupled through gap junction channels, which allow ionic current to spread intercellularly from one cell to the next. In addition, it is possible that cardiac cells are coupled through the electric potential in the junctional cleft space between neighboring cells. We develop and analyze a mathematical model of two cells coupled through a common junctional cleft potential. Consistent with more detailed models, we find that the coupling mechanism is highly parameter dependent. Analysis of our model reveals that there are two time scales involved, and the dynamics of the slow subsystem provide new mathematical insight into how the coupling mechanism works. We find that there are two distinct types of propagation failure and we are able to characterize parameter space into regions of propagation success and the two different types of propagation failure.     

Fractional contribution of major ions to the membrane potential of Drosophila melanogaster oocytes

In ovarian follicles of Drosophila melanogaster, ion substitution experiments revealed that K⁺ is the greatest contributor (68%) in setting oocyte steady‐state potential (E_m), while Mg²⁺ and a metabolic component account for the rest. Because of the intense use made of Drosophila ovarian follicles in many lines of research, it is important to know how changes in the surrounding medium, particularly in major diffusible ions, may affect the physiology of the cells. The contributions made to the Drosophila oocyte membrane potential (E_m) by [Na⁺]_o, [K⁺]_o, [Mg²⁺]_o, [Ca²⁺]_o, [Cl^?]_o, and pH (protons) were determined by substitutions made to the composition of the incubation medium. Only K⁺ and Mg²⁺ were found to participate in setting the level of E_m. In follicles subjected to changes in external pH from the normal 7.3 to either pH 6 or pH 8, E_m changed rapidly by about 6 mV, but within 8 min had returned to the original E_m. Approximately half of all follicles exposed to reduced [Cl^?]_o showed no change in E_m, and these all had input resistances of 330 kΩ or greater. The remaining follicles had smaller input resistances, and these first depolarized by about 5 mV. Over several minutes, their input resistances increased and they repolarized to a value more electronegative than their value prior to reduction in [Cl^?]_o. Together, K⁺ and Mg²⁺ accounted for up to 87% of measured steady‐state potential. Treatment with sodium azide, ammonium vanadate, or chilling revealed a metabolically driven component that could account for the remaining 13%. © 2009 Wiley Periodicals, Inc.