An active electrical response in fibroblasts

L cells have a resting potential of about -16 mv (internal negative) at 37°C in Dulbecco''s modified Eagle''s medium containing 10% fetal calf serum and a potassium concentration of 5.4 mM. Membrane resistivity is about 20,000 Ωcm² when the surface filopodia described by others are taken into account. Mechanical and electrical stimuli can evoke an active response from mouse L cells, cells of the 3T3 line, and normal fibroblasts which we have termed hyperpolarizing activation or the H.A. response. This consists of a prolonged (3–5 sec) increase in the membrane permeability by a factor of 2–10 with a parallel increase in membrane potential to about -50 mv. The reversal potential for the H.A. response is -80 mv. The resting cells are depolarized to about -12 mv when the external medium contains 27 mM potassium, and the potential reached at the peak of the H.A. response is about -30 mv. The reversal potential for the H.A. response is about -40 mv in 27 mM external potassium. This effect of potassium ions on the reversal potential of the H.A. response leads us to conclude that the response represents an increase in membrane permeability, predominantly to potassium, by at least a factor of five. This increase must be greater than 20-fold if previous measurements of the ratio of potassium permeability to chloride permeability in L cells are valid for the preparation used in the present study.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

Membrane Potentials of the Lobster Giant Axon Obtained by Use of the Sucrose-Gap Technique

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.     

The effects of some ions on the membrane potential of the giant axon of Myxicola

The effects of various ions on the resting membrane potential of the giant axons of Myxicola were determined. The mean resting potential in artificial sea water is 69 mv, inside negative. The membrane potential decreases with incresing external potassium concentrations, while changes in the sodium and chloride concentrations have little or no effect. For potassium concentrations greater than 50 mM/L the relation between membrane potential and concentration approximates that of a perfectly selective potassium electrode. The data for the whole range of concentrations examined can be well fitted by the equation: It was pointed out that the Myxicola giant axons can be studied under space voltage clamp and can be made available in the laboratory for 12 months out of the year. Myxicola then should become a very useful preparation for the study of membrane phenomena.     

Electrical Properties of Neurospora crassa : Effects of external cations on the intracellular potential

Glass micropipette electrodes have been employed to study the transsurface potential difference of Neurospora crassa. For mature hyphae grown in agar cultures, the internal potential is large and negative, often exceeding -200 mv. The potential is sensitive to the concentrations of extracellular potassium, sodium, hydrogen, and calcium ions, but does not vary in a manner which is readily explained by ionic diffusion potentials. With extracellular solutions containing only potassium chloride (or sulfate) and sucrose, the internal potential shifts toward zero (becomes less negative) at 45 mv per tenfold increase of potassium, over the range 0.1 to 10 mM. A similar result has been found with sodium, though the slope is only 33 mv/log unit. Calcium (1 mM) diminishes the influence of potassium and sodium by 60 to 70 per cent. As potassium or sodium is raised above 20 mM, the slope of the internal potential increases sharply to 85 to 90 mv/log unit, both in the presence and absence of calcium. With increasing hydrogen ion concentration, too, the internal potential shifts toward zero; in this case the slope is about 12 mv/pH unit at pH 9 and rises smoothly to 33 mv/pH unit at pH 3. All these phenomena are probably properties of the plasma membrane. The polysaccharide cell wall contains few fixed negative charges, has a low transverse resistance, and supports very little potential difference when separated from the plasma membrane.     

Effect of External and Internal pH Changes on K and Cl Conductances in the Muscle Fiber Membrane of a Giant Barnacle

The membrane potential and conductance of the giant muscle fiber of a barnacle (Balanus nubilus Darwin) were analyzed in relation to changes in the external (3.5–10.0) and the internal (4.7–9.6) pH, under various experimental conditions. A sharp increase in membrane conductance, associated with a large increase in conductance to Cl ions, was observed when the external pH was lowered to values below 5.0. The ratio of Cl to K conductance in normal barnacle saline is between –1/7 at pH 7.7, whereas at pH 4.0 the ratio is about 6–9. The behavior of the membrane in response to pH changes in a Cl-depleted muscle fiber shows that the K conductance decreases with decreasing external pH for the whole range of pH examined. A steep increase in Cl conductance is also observed when the internal pH of the fiber is lowered below 5.0. The K to Cl conductance ratio increases with increasing internal pH in a manner very similar to that found when the external pH is raised above 5.0. These facts suggest that the membrane is amphoteric with positive and negative fixed charge groups having dissociation constants such that at pH greater than 5, negative groups predominate and cations permeate more easily than anions, while at lower pH positive groups predominate, facilitating the passage of anions through the membrane.     

Relative Ion Permeabilities in the Crayfish Giant Axon Determined from Rapid External Ion Changes

The changes in membrane potential of isolated, single crayfish giant axons following rapid shifts in external ion concentrations have been studied. At normal resting potential the immediate change in membrane potential after a variation in external potassium concentration is quite marked compared to the effect of an equivalent chloride change. If the membrane is depolarized by a maintained potassium elevation, the immediate potential change due to a chloride variation becomes comparable to that of an equivalent potassium change. There is no appreciable effect on membrane potential when external sodium is varied, at normal or at a depolarized membrane potential. Starting from the constant field equation, expressions for the permeability ratios P _Cl/P _K, P _Na/P _K, and for intracellular potassium and chloride concentrations are derived. At normal resting membrane potential, P _Cl/P _K is 0.13 but at a membrane potential of -53 mv (external potassium level increased about five times) it is 0.85. The intracellular concentrations of potassium and chloride are estimated to be 233 and 34 mM, respectively, and it is pointed out that this is not compatible with ions distributed in a Nernst equilibrium across the membrane. It is also stressed that the information given by a plot of membrane potential vs. the logarithm of external potassium concentrations is very limited and rests upon several important assumptions.     

The control of membrane ionic currents by the membrane potential of muscle

Comparisons between electrotronic potentials and certain predicted curves allow the identification of the membrane potential at which the sodium and potassium currents are switched on in frog sartorius. The activation potentials (the membrane potentials at which the ionic currents are great enough to be resolved by the method) are functions of the resting potential and time but not of ionic concentration. In the normal fiber, the activation potential for sodium lies nearer the resting potential and depolarizations set off sodium currents and action potentials. Below a resting potential of 55 to 60 mv. sodium activation is lost and conduction is impossible. A tenfold increase of calcium concentration lowers (moves further from the resting potential) the sodium activation potential by 20 to 25 mv. whereas the potassium activation potential is lowered by only 15 mv. Certain consequences of this are seen in the behavior of the muscle cell when it is stimulated with long duration shock.     

Calcium and action potentials of bullfrog sympathetic ganglion cells

Bullfrog sympathetic ganglion cells were capable of producing action potentials (Ca spikes) in an isotonic (84 mM) CaCl2 solution. The peak level of Ca spikes showed an approximately 30 mv increase with a 10-fold increase in the Ca concentration. Na as well as Ca ions were capable of acting as charge carriers during the production of action potentials in a solution containing relatively high Ca and relatively low Na ions. A decrease in the external Ca concentration depressed the maximum rate of rise at a fixed resting potential level, and increased the maximum rate of rise of the Na spikes at a high resting potential level at which Na inactivation was completely depressed. Compared to Na spikes, Ca spikes were less sensitive to TTX and procaine. Ganglion cells were also capable of producing action potentials (Sr spikes) in an isotonic SrCl₂ solution and prolonged action potentials in an isotonic BaCl₂ solution, but these cells were rendered inexcitable in an isotonic MgCl₂ solution. The peak level of the Sr spikes was dependent on the external Sr concentration and was insensitive to both TTX and procaine. Sr ions, like Ca ions, reduced Na inactivation during the resting state, and depressed the maximum rate of rise of the Na spikes at a high resting potential level. It was concluded that Ca (and Sr) ions exert dual actions on the membrane; namely, regulating the Na permeability and acting as charge carriers during the active state of the membrane.     

Two Light-Induced Processes in the Photoreceptor Cells of Limulus Ventral Eye

The dark-adapted current-voltage (I-V) curve of a ventral photoreceptor cell of Limulus, measured by a voltage-clamp technique, has a high slope-resistance region more negative than resting voltage, a lower slope-resistance region between resting voltage and zero, and a negative slope-resistance region more positive than 0 v. With illumination, we find no unique voltage at which there is no light-induced current. At the termination of illumination, the I-V curve changes quickly, then recovers very slowly to a dark-adapted configuration. The voltage-clamp currents during and after illumination can be interpreted to arise from two separate processes. One process (fast) changes quickly with change in illumination, has a reversal potential at +20 mv, and has an I-V curve with positive slope resistance at all voltages. These properties are consistent with a light-induced change in membrane conductance to sodium ions. The other process (slow) changes slowly with changes in illumination, generates light-activated current at +20 mv, and has an I-V curve with a large region of negative slope resistance. The mechanism of this process cannot as yet be identified.     

Relationship between ion distribution and membrane potential during a steady state

Equations were derived showing the relationship between the membrane potential and the quantities which influence it under steady state conditions. Essentially, the membrane potential is caused by the valence and concentration of the non-permeating ions. The permeating ions can modify the membrane potential by altering the relative concentration of the non-permeating ions with respect to the concentration of the permeating ions. For muscle, the sodium cations act as the non-permeating ions in the extracellular environment by the maintenance of some type of active metabolic process and large anions act as the non-permeating ions in the intracellular environment. Both of these non-permeating ions contribute about equally to the maintenance of the resting membrane potential. When the active metabolic process for sodium extrusion breaks down or when acids are added, the membrane potential should decrease. Water should enter the cell when the sodium metabolic process is diminished; water should leave the cell when acids are added. When acid is added, it is expected that the cations potassium and sodium will leave the cell with little or no shift of the chloride ions.     

Some Electrical Properties of a Nuclear Membrane Examined with a Microelectrode

Electrical potential and resistance were measured with microelectrodes in in situ and isolated nuclei of gland cells of Drosophila flavorepleta. The nucleus-cytoplasm boundary was found to be rather impermeable to ion diffusion. It presents a resistance of the order of 1 Ω cm² and sustains a "resting" potential, the nucleoplasm being about 15 mv negative with respect to the cytoplasm. Both the resistance and potential appear to be associated with the nuclear membrane: the potential declines to zero and the resistance to a fraction of its original value, when the membrane is perforated experimentally.     

The Effects of Various Ions on Resting and Spike Potentials of Barnacle Muscle Fibers

Effects of monovalent cations and some anions on the electrical properties of the barnacle muscle fiber membrane were studied when the intra- or extracellular concentrations of those ions were altered by longitudinal intra-cellular injection. The resting potential of the normal fiber decreases linearly with increase of logarithm of [K⁺]_out and the decrement for a tenfold increase in [K⁺]_out is 58 mv when the product, [K⁺]_out ·[Cl^-]_out, is kept constant. It also decreases with decreasing [K⁺]_in but is always less than expected theoretically. The deviation becomes larger as [K⁺]_in increases and the resting potential finally starts to decrease with increasing [K⁺]_in for [K⁺]_in > 250 mM. When the internal K⁺ concentration is decreased the overshoot of the spike potential increases and the time course of the spike potential becomes more prolonged. In substituting for the internal K⁺, Na⁺ and sucrose affect the resting and spike potentials similarly. Some organic cations (guanidine, choline, tris, and TMA) behave like sucrose while some other organic cations (TEA, TPA, and TBA) have a specific effect and prolong the spike potential if they are applied intracellularly or extracellularly. In all cases the active membrane potential increases linearly with the logarithm of [Ca⁺⁺]_out/[K⁺]_in and the increment is about 29 mv for tenfold increase in this ratio. The fiber membrane is permeable to Cl^- and other smaller anions (Br^- and I^-) but not to acetate^- and larger anions (citrate^-, sulfate^-, and methanesulfonate^-).     

Origin of Axon Membrane Hyperpolarization under Sucrose-Gap

One of the disadvantages of the sucrose-gap method for measuring membrane potentials with extracellular electrodes is a membrane hyperpolarization of the order of 30 to 60 mv, as compared with the resting potential obtained with intracellular microelectrodes in the absence of a sucrose-gap. In the present study the contribution of the sucrose-sea water junction potential to this hyperpolarization effect has been evaluated by comparing the effects on the resting potential of several anion and cation substitutions in the sea water bathing the lobster giant axon under sucrose-gap. Measurements with microelectrodes demonstrate a significant liquid junction potential between sucrose and standard artificial sea water. The value of this liquid junction potential as well as the measured resting membrane potential varies as a function of the anions and cations substituted in the sea water. Both the liquid junction potential and the sucrose-gap-induced hyperpolarization can be eliminated by using a low mobility anion to replace most of the chloride in sea water while the normal cation content remains unchanged. These data provide evidence that loop currents at the sucrose-sea water-axon junctions are at least partly responsible for membrane hyperpolarization under a sucrose gap.     

Anomalous proton selectivity in a large channel: colicin A

Some of the bactericidal proteins known as colicins exert their toxic action by forming a large, nonselective channel in the inner membrane of target bacteria. The structure of this channel is unknown. It conducts large ions but has a much smaller conductance than would be expected for a channel of its deduced size. Here we report that the colicin channel, particularly the colicin A channel, is selective for protons over other cations (and anions) by many orders of magnitude. This was deduced from measurements of reversal potentials in pH gradients across planar lipid bilayers containing these channels. For example, in symmetric 0.1 M KCl with a pH 5/pH 8 gradient across the membrane, the reversal potential of colicin A is -21 mV, rather than 0. Such a result would be unremarkable for a narrow channel but is beyond explanation by current understanding of permeation for a channel of its diameter. For this reason, we re-examined the issue of the diameter of the channel lumen and confirmed that the lumen is indeed "too large" ( approximately 10 A) to select for protons by the amount that we measure. We are thus compelled to propose that an unorthodox mechanism is at work in this protein.     

Chloride fluxes in isolated dialyzed barnacle muscle fibers

Chloride outflux and influx has been studied in single isolated muscle fibers from the giant barnacle under constant internal composition by means of a dialysis perfusion technique. Membrane potential was continually recorded. The chloride outfluxes and influxes were 143 and 144 pmoles/cm²-sec (mean resting potential: 58 mv, temperature: 22°–24°C) with internal and external chloride concentrations of 30 and 541 mM, respectively. The chloride conductance calculated from tracer measurements using constant field assumptions is about fourfold greater than that calculated from published electrical data. Replacing 97% of the external chloride ions by propionate reduces the chloride efflux by 51%. Nitrate ions applied either to the internal or external surface of the membrane slows the chloride efflux. The external pH dependence of the chloride efflux follows the external pH dependence of the membrane conductance, in the range pH 3.9–4.7, increasing with decreasing pH. In the range pH 5–9, the chloride efflux increased with increasing pH, in a manner similar to that observed in frog muscle fibers. The titration curve for internal pH changes in the range 4.0–7.0 was quantitatively much different from that for external pH change, indicating significant asymmetry in the internal and external pH dependence of the chloride efflux.     

Electrical Characteristics and Activation Potential of Bufo Eggs

Electrical characteristics and their changes during activation were studied with the microelectrodes on the oocytes and eggs of the toad, Bufo vulgaris formosus Boulenger. In young oocytes, the membrane characteristics had some similarities to those of nerve and muscle, except for a relatively large resistance of 25 KΩcm.² and an absence of the action potential in the former. After maturation, however, the membrane characteristics became entirely different from those of oocytes and other excitable tissues. In the mature eggs the membrane resistance was measured to be as high as 200 KΩcm.², and no specific permeability of the membrane to potassium ions was observable. A slow monophasic change in the membrane potential was recorded in every activation produced by mechanical stimulation, and termed "activation potential." In fresh water, its amplitude was as large as 80 to 90 mv. with an overshoot of about 50 mv. The activation potential might be comparable to the action potential of nerve and muscle, but was fundamentally different in ionic mechanism from the latter, since the former was caused by a marked increase in permeability to chloride ions.     

Influence of Some Ions on the Membrane Potential of Ascaris Muscle

The influence of several ions on the membrane potential of the somatic muscle of Ascaris has been investigated by changing their concentration in the surrounding solution. When [K]_o is increased at the expense of [Na]_o leaving [Cl]_o constant, the membrane potential is first seen to increase. [K]_o higher than 45 mM reduces the membrane potential with a slope of 23 mv for a tenfold change in [K]_o. However, when [K]_o is increased keeping [Na]_o and [Cl]_o low and constant, the line relating the membrane potential with log [K]_o has a slope of almost 50 mv. If [Cl]_o is reduced in the absence of external Na, after the [K]_o is increased to 45 mM, the membrane potential decreases with a slope of 59 mv per tenfold change in [Cl]_o in close agreement with the Nernst equation. If Cl^- is replaced by SO₄ ^2-, a depolarization is produced, while chloride replacement by NO₃ ^-, Br^-, and I^- results in a hyperpolarization of the membrane. Removal of the external Na⁺ ions increases the average membrane potential by 17 mv.     

The relations between prepotential,resting potential,and latent period in frog muscle fibers

1. Prepotentials and action potentials were recorded from amphibian striated muscle fibers. Intracellular electrodes were used for stimulating and recording. The resting potential was varied from 55 to 120 mv. by alterations of the KCl concentration of the Ringer's fluid. The magnitude of the prepotential at the initiation of the spike potential was measured and compared to the resting potential and the latent period (time between stimulus "make" and excitation). The magnitude of this prepotential varied with the resting potential. 2. A large prepotential or cathodal depolarization was required to excite a fiber with a high resting potential. If a fiber with a high resting potential fired late (long latency), the adequate prepotential was larger than if the fiber fired early. Fibers with low resting potentials had smaller adequate prepotentials. Also, the adequate prepotential was independent of the latent period, in these depolarized fibers. 3. If the concentration of Ca⁺⁺ was increased tenfold, the adequate prepotential of depolarized fibers became strongly dependent upon the latency. 4. Fibers with large or normal resting potentials were prone to respond repetitively during the passage of long duration shock, whereas depolarized and Ca⁺⁺-treated fibers were not. 5. The so-called critical membrane potential (which is defined as the transmembrane potential at the point of excitation) was not independent of the resting potential.     

Effect of acetylcholine on postjunctional membrane permeability in eel electroplaque

Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na+ was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of Cl- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na+, Ca++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation.