The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

The Inhibition of Sodium Currents in Myelinated Nerve by Quaternary Derivatives of Lidocaine

The inhibition of sodium currents by quaternary derivatives of lidocaine was studied in single myelinated nerve fibers. Membrane currents were diminished little by external quaternary lidocaine (QX). QX present in the axoplasm (<0.5 mM) inhibited sodium currents by more than 90%. Inhibition occurred as the sum of a constant, tonic phase and a variable, voltage-sensitive phase. The voltage-sensitive inhibition was favored by the application of membrane potential patterns which produce large depolarizations when sodium channels are open. Voltage-sensitive inhibition could be reversed by small depolarizations which opened sodium channels. One explanation of this observation is that QX molecules enter open sodium channels from the axoplasmic side and bind within the channels. The voltage dependence of the inhibition by QX suggests that the drug binds at a site which is about halfway down the electrical gradient from inside to outside of the sodium channel.     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Diffusion of Ions in Myelinated Nerve Fibers

The diffusion of ions towards or away from the inner side of the nodal membrane in preparations, the cut ends of which are placed in various media, was investigated. The ion concentration changes were calculated by numerical solution of the unidimensional electrodiffusion equation under a variety of media compositions, axoplasmic diffusion coefficients, and internal anionic compositions. The potassium and cesium ion diffusion along the axon towards the node was determined experimentally by two different electrophysiological methods. On the basis of comparison between the experimental data and the computational predictions the axoplasmic potassium ion diffusion coefficient was determined to be almost equal to that in free aqueous solution, while that of cesium ion was close to one half of that in aqueous solution. Utilizing the values of diffusion parameters thus determined, we solved the electrodiffusion equation for a number of common experimental procedures. We found that in short fibers, cut 0.1-0.2 cm at each side of the node, the concentration approached values close to the new steady-state values within 5-30 min. In long fibers (over 1 cm long) steady-state concentrations were obtained only after a few hours. Under some conditions the internal concentrations transiently overshot the steady-state values. The diffusion potentials generated in the system were also evaluated. The ion concentration changes and generation of diffusion potential cannot be prevented by using side pools with cation content identical to that of the axoplasm.     

Effects of Permeant Cations on K+ Channel Gating in Nerve Axons Revisited

An increase in extracellular potassium ion concentration, K _o , significantly slows the potassium channel deactivation rate in squid giant axons, as previously shown. Surprisingly, the effect does not occur in all preparations which, coupled with the voltage independence of this result in preparations in which it does occur, suggests that it is mediated at a site outside of the electric field of the channel, and that this site is accessible to potassium ions in some preparations, but not in others. In other words, the effect does not appear to be related to occupancy of the channel by potassium ions. This conclusion is supported by a four-barrier, three-binding site model of single file diffusion through the channel in which one site, at most, is unoccupied by a potassium ion (single-vacancy model). The model is consistent with current-voltage relations with various levels of K _o , and, by definition, with multiple occupancy by K⁺. The model predicts that occupancy of any given site is essentially independent of K _o (or K _i ). The effects of extracellular Rb⁺ and Cs⁺ on gating are strongly voltage dependent, and they were observed in all preparations investigated. Consequently, the mechanism underlying these results would appear to be different from that which underlies the effect of K⁺ on gating. In particular, the effect of Rb⁺ on gating is reduced by strong hyperpolarization, which in the context of the occupancy hypothesis, is consistent with the voltage dependence of the current-voltage relation in the presence of Rb⁺. The primary, novel, finding in this study is that the effects of Cs⁺ are counterintuitive in this regard. Specifically, the slowing of channel deactivation rate by Cs⁺ is also reduced by hyperpolarization, similar to the Rb⁺ results, whereas blockade is enhanced, which is seemingly inconsistent with the concept that occupancy of the channel by Cs⁺ underlies the effect of this ion on gating. This result is further elucidated by barrier modeling of the current-voltage relation in the presence of Cs⁺. Received: 19 December 1995/Revised: 10 June 1996     

Permeability of Lipophilic Cations

The permeability (P) of a lipophilic cation, triphenylmethylphosphonium(TPMP⁺) which is frequently used as a membrane potential probe,has been measured in Chara australis (Charophyceae). P_TPMP+across biological membranes is usually thought to be very highbut this is not the case across the plasmalemma of Chara. Thepermeability of TPMP⁺ across the plasmalemma was found to betypical of inorganic cations, about 1.0 nm s^–1. Estimateswere made of the permeability of lipophilic cations across someother cell membranes, based on previously published work. Thepermeability of TPMP⁺ across the plasma membranes of the redalga, Griffithsia monilis and the blue-green alga, Anabaenavariabilis was about 2–5 nm s^–1. The permeabilityof TPMP⁺ across the plasma membranes of eukaryotes and prokaryotesappears to be similar. The permeability of lipophilic cationsacross the cristae of isolated mitochondria are exceptionallyhigh, about 170 nm s^–1. TPMP⁺ did not behave as a thiamineanalogue in Chara, unlike in the case of yeast. The means ofentry of TPMP⁺ into the Chara cell, driven by the electrochemicalgradient across the plasmalemma, has not been identified. Thepresence of a second lipophilic cation probe, DDA⁺ (dibenzyldimethylammonium),caused a decrease in the uptake flux of TPMP⁺; this suggeststhat the two lipophilic cations compete for the same site atthe surface of the plasmalemma. Key words: Chara australis, TPMP⁺, Permeability, Lipophilic cation     

Potassium Channels in Myelinated Nerve : Selective permeability to small cations

The permeability of K channels to various cations is studied in myelinated nerve. Ionic currents under voltage clamp are measured in Ringer solution containing tetrodotoxin and a high concentration of the test ion. Reversal potentials for current in K channels are determined and used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The ratios P_Tl:P_K:P_Rb:P_NHNH4 are 2.3:1.00:0.92:0.13. No other ions are found to be measurably permeant including Li⁺, Na⁺, Cs⁺, methylamine, guanidine, hydrazine, or hydroxylamine. The ratio P_Na/P_K is less than 0.01. Potassium conductance is depressed at pH values below 5.0. Leakage conductance is higher in K, Rb, Cs, NH₄, and Tl Ringer than in Na Ringer, but the selectivity sequence probably is not the same as for K channels. The hypothesis is offered that the narrowest part of the K channel is a circle of oxygen atoms about 3 Å in diameter with low electrostatic field strength.     

侧足厚蟹有髓鞘神经纤维的超微结构电镜观察

侧足厚蟹(Helice latimera)的有髓鞘神经纤维直径约在5—12μm之间,髓鞘厚约2μm左右。髓鞘由内外膜层和中间微管层组成。外膜层层数约20层,排列紧密,微管层厚约0．31μm,内膜层层数约20层。轴突膜形成嵴,伸进轴腔内,在轴腔内还存在维管束和膜层束结构。在髓鞘结构中观察到两种类型的高嗜锇性区域,一种是由排列规则且明暗交替的膜层结构组成,另一种是非膜层结构组成。     

有髓轴突横断损伤后钠通道聚集状态研究

目的:研究有髓轴突横断损伤后郎飞结区钠通道聚集状态的变化.方法:用雪旺细胞-背根神经元髓鞘化共培养系统复制周围神经髓鞘形成和郎飞结发育,于髓鞘化培养基中共培养第14天用前房角切开刀造成有髓轴突横断损伤,在损伤后1、2、3、4、5、6、7、14天进行髓鞘碱性蛋白和钠通道免疫荧光染色,损伤前共培养作为对照.利用SPOT图像分析软件测量钠通道聚集簇的直径、长度和直径/长度比.结果:损伤前钠通道蛋白在有髓轴突郎飞结区形成直径/长度比略大于1的聚集簇;有髓轴灾横断损伤后钠通道蛋白沿轴突纵向扩散,钠通道聚集簇的直径/长度比逐渐减小,损伤后第14天已无法检测到钠通道表达.损伤区出现节段性脱髓鞘.结论:轴突横断损伤可造成钠通道聚集簇扩散、消失,导致郎飞结结构破坏.     

Effects of Divalent Cations,Trypsin, and Phospholipases on the Passive Permeability to Sodium of Inside-Out Vesicles From Human Red Cells

Inside-out vesicles (IOV) were prepared from human red blood cells. Steady-state uptake of ²²Na was observed to generally follow an exponential time course with a rate constant of 1.57 ± 0.09 h^?1 (SE). One week of cold storage (0–4°C) increased the rate constant to 2.50 ± 0.12 h ^?1 (SE). Mg²⁺, Ca²⁺, or Sr²⁺ decreased the rate of ²²Na uptake with no observable differences between the three divalent cations when tested at concentrations of 50 μM. Mg²⁺ was shown to decrease the rate of ²²Na uptake at concentrations as low as 5 μM with maximal effect at 50 to 100 μM. The decrease in rate of ²²Na uptake induced by Mg²⁺ could be enhanced by exposure of IOV to Mg²⁺ for longer periods of time. Trypsin treatment of IOV increased the rate of uptake of ²²Na and was dependent on the concentration of trypsin added between 5 to 25 μg/ml (treated for 5 min at 25°C). The ability of Mg²⁺ (50 μM) to decrease the rate of ²²Na uptake was still observed after maximal trypsin treatment. Phospholipase A₂ or phospholipase C treatment of IOV increased the rate of ²²Na uptake and was dependent on the amount of phospholipase A₂ (0.1 to 1.0 units/ml) or phospholipase C (0.25 to 2.5 units/ml) added (treated for 5 min at 25°C). After phospholipase A₂ treatment, the observed decrease in the rate of ²²Na uptake induced by Mg²⁺ (50 μM) was generally greater than controls. After phospholipase C treatment, the observed decrease in rate of ²²Na uptake induced by Mg²⁺ (50 μM) was less or absent when compared with controls. Phospholipase C treatment was less effective in preventing the Mg²⁺ effect the longer IOV were exposed to Mg²⁺. The results suggest that Mg²⁺ binds to phospholipid head-groups to reduce Na permeability perhaps by inducing a change in bilayer structure or phospholipid association.     

He-Ne激光照射罗氏沼虾腹髓鞘神经纤维的电镜观察

用He Ne激光照射罗氏沼虾 (Macrobrachiumrosenbergii)的腹髓鞘神经纤维 ,结果表明 ,神经胶质细胞为不规则形 ,核的一端较大 ,另一端分叉 ,半包裹着轴突 ,呈蝴蝶形 ,核的横轴直径为 3 0 7μm ,轴突直径为 1 5 3 μm ;在髓鞘中普遍出现增厚的膜层结构 ,且膜层结构厚薄不一 ,具有高嗜锇性、电子密度大 ,正常的膜层厚度为 3 3 3 3nm ,增厚的膜层厚度可达 1 0 0nm。在两膜层之间存在着许多颗粒状物质。     

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of “prototypical” Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.     

Computation of Impulse Initiation and Saltatory Conduction in a Myelinated Nerve Fiber

A mathematical model of the electrical properties of a myelinated nerve fiber is given, consisting of the Hodgkin-Huxley ordinary differential equations to represent the membrane at the nodes of Ranvier, and a partial differential cable equation to represent the internodes. Digital computer solutions of these equations show an impulse arising at a stimulating electrode and being propagated away, approaching a constant velocity. Action potential curves plotted against distance show discontinuities in slope, proportional to the nodal action currents, at the nodes. Action potential curves plotted against time, at the nodes and in the internodes, show a marked difference in steepness of the rising phase, but little difference in peak height. These results and computed action current curves agree fairly accurately with published experimental data from frog and toad fibers.     

Laser Temperature-Jump Technique for Relaxation Studies of the Ionic Conductances in Myelinated Nerve Fibers

A temperature-jump technique for single nodes of Ranvier has been developed using a pulsed laser system. The temperature perturbation was accomplished by firing the laser beam obtained from a neodymium rod through the solution surrounding a single node. The temperature step was achieved within 1 msec using the laser in the normal mode of operation. During the voltage-clamped steady-state current a temperature jump from 4°C increased the current to a new steady-state value within the time course of the T-jump. This finding suggests that the maximum potassium permeability P_K has a rapid relaxation time and that the steady-state value of n (the value of potassium permeability divided by its maximum value) is relatively independent of temperature. T-jumps applied during the voltage-clamped sodium currents showed that the sodium permeability changed with a relaxation time that was also shorter than the duration of the normal mode laser output. T-jumps observed during a hyperpolarization or at the resting potential showed no detectable conductance change. When a T-jump immediately preceded a voltage clamp pulse the technique was then used to investigate the effect of changes in the steady-state temperature on the ionic conductances. It was found that the magnitude of the change in membrane current due to a T-clamp was directly related to the level of cathodal polarization.     

The Functional Surface Charge Density of a Fast K Channel in the Myelinated Axon of Xenopus laevis

The action of Mg²⁺ on the putative xKv1.1 channel in the myelinated axon of Xenopus laevis was analyzed in voltage clamp experiments. The main effect was a shift in positive direction of the open probability curve (16 mV at 20 mm Mg²⁺), calculated from measurements of the instantaneous current at Na reversal potential after 50–100 msec steps to different potentials. The shift was measured at an open probability level of 25% to separate it from shifts of other K channel populations in the nodal region. The results could be explained in terms of screening effects on fixed charges located on the surface of the channel protein. Using the Grahame equation the functional charge density was estimated to −0.45 e nm⁻². Analyzing this value, together with previously estimated values from other K channels, with reference to the charge of different extracellular loops of the channel protein, we conclude that the loop between the transmembrane S5 segment and the pore forming P segment determines the functional charge density of voltage-gated K channels. Received: 11 December 1997/Revised: 24 April 1998     

Effect of Cations on Effective Permeability of Leaf Cuticles to Sulfuric Acid

Many plants are exposed to prolonged episodes of anthropogenic acid precipitation with pH values of 4 or less, but there is little evidence of widespread direct damage to the plant cells. Acids appear to permeate leaf cuticle via charged pores, which act as a fixed buffer that delays but does not stop acid movement. We investigated the effect of cations on the movement of protons through astomatous isolated leaf cuticles of pear (Pyrus communis L.) and rough lemon (Citrus limon [L.] Burm. fils cv Ponderosa). Chloride salt solutions of Na, K, Ca, Cd, Mg, Gd, or Y in a diffusion apparatus were applied to the morphological inner surface of the cuticle, while the outer surface faced a large volume of pH 3 or 4 sulfuric acid. Effective permeability was calculated from the change in the pH of the inner solution as measured with a pH microelectrode. Monovalent cations caused either no change (pear) or promotion (rough lemon) of proton movement. Divalent cations reduced proton movement in a concentration-dependent manner (both species), whereas trivalent cations (rough lemon only) caused the effective permeability to decrease to near zero. Inhibition by 10 mM CaCl2 was reversed with water. The effects of these cations on the permeability of cuticles to protons was used to elucidate mechanisms by which cations can protect leaves from acid precipitation in nature.     

Induction of Morphogenesis in Geodermatophilus by Inorganic Cations and by Organic Nitrogenous Cations

Morphogenesis of Geodermatophilus strain 22-68 involves two stages, a motile rod (R form) and an irregularly shaped cluster of coccoid cells (C form). A variety of mono- and divalent cations have been found to induce R-form to C-form morphogenesis and to maintain the organism in the C form. Concentration optima for all cations exceeded 100 mM. Results indicated that uptake of cations was accompanied by extrusion of intracellular protons, causing an increase in intracellular pH. A variety of organic amines also induced morphogenesis. Organic amines were taken up in the dissociated free base form, causing the intracellular pH to rise. None of these compounds was utilized as a carbon or nitrogen source.