Inconsistent sodium current records derived on Ranvier nodes with a commercially available potential clamp device according to Nonner

We tried to reproduce some basic implications of the Hodgkin-Huxley-Frankenhaeuser formalism by measuring sodium currents in single myelinated nerve fibres with a commercially available version of the potential clamp device according to Nonner. The following contradictory observations were made: 1. The potential dependence of the time to peak sodium currents showed a discontinuity around the sodium equilibrium potential. 2. Defining the sodium permeability PNa by the constant field equation and fitting the peak PNa-voltage relation by a sigmoid function we obtained unbelievable high values of PNa at rest. 3. Testing PNa as calculated by the constant field equation by so-called "sodium tail current" experiments we obtained instantaneous changes of PNa. Summing up, neither the kinetics of sodium currents nor the constant field concept as tested with the equipment used seem to agree satisfactorily with the standard data of sodium currents in Ranvier nodes.     

High standard one-loop potential clamp device for Ranvier nodes

Starting from the observation that using a conventional potential clamp device for membrane current measurements in Ranvier nodes neither the kinetics of sodium currents nor the constant field concept agree satisfactorily with the Hodgkin-Huxley-Frankenhaeuser (HHF)-formalism, an extendend measuring system has been developed. The extensions introduced base largely on physical implications of myelinated nerve fibres which give rise 1. to systematic distortions of any current records at the high frequency end and 2. to current proportional deviations of the membrane potential from desired potential values. In addition, we provided to meet any unwanted current load during membrane current measurements and to push the time resolution of the measuring system to the highest possible value. After having tested thoroughly the new circuitry by appropriate physical methods, from sodium current measurements the following conclusions were drawn: 1. Occasional deviations of sodium current kinetics near the sodium equilibrium potential from the predictions of the HHF-formalism are measurement errors. 2. The constant field formalism holds for sodium currents in the potential range of biological relevance only. 3. Instantaneous sodium current measurements, however, are of unsatisfactory significance because for this kind of experiments the time resolution of the measuring system used might be still too low.     

Block of single cardiac sodium channels by intracellular magnesium

Currents through single cardiac sodium channels have been measured in inside-out patches from guinea pig ventricular cells. To abolish the fast inactivation, Na channels were modified by DPI 201–106. In symmetrical Na solutions, a diminution of outward sodium currents can be observed that depends on the intracellular magnesium concentration and the membrane potential. Inward currents were not altered by the concentrations of magnesium used (between 0 and 22.5 mmol/1). In Mg free solutions a linear current-voltage relation can also be measured in the range of outward Na currents. At +60 mV (symmetrical Na solutions, single channel conductance 24 pS) a half maximal block of cardiac Na channels by intracellular magnesium was found at 2.1 mmol/l. From the analysis of single channel current-voltage relationships the concentration and voltage-dependent block by intracellular magnesium of cardiac sodium channels could be described as binding of Mg at one site with a K _d value of 5.1 mmol/1 at 0 mV. The site is located at an electrical distance of 0.18 from the inside. Offprint requests to: B. Nilius     

Magnitude and location of surface charges on Myxicola giant axons

The effects of changes in the concentration of calcium in solutions bathing Myxicola giant axons on the voltage dependence of sodium and potassium conductance and on the instantaneous sodium and potassium current-voltage relations have been measured. The sodium conductance-voltage relation is shifted along the voltage axis by 13 mV in the hyperpolarizing direction for a fourfold decrease in calcium concentration. The potassium conductance-voltage relation is shifted only half as much as that for sodium. There is no effect on the shape of the sodium and potassium instantaneous current-voltage curves: the normal constant-field rectification of potassium currents is maintained and the normal linear relationship of sodium currents is maintained. Considering that shifts in conductances would reflect the presence of surface charges near the gating machinery and that shape changes of instantaneous current-voltage curves would reflect the presence of surface charges near the ionic pores, these results indicate a negative surface charge density of about 1 electronic charge per 120 A2 near the sodium gating machinery, about 1 e/300 A2 for the potassium gating machinery, and much less surface charge near the sodium or potassium pores. There may be some specific binding of calcium to these surface charges with an upper limit on the binding constant of about 0.2 M-1. The differences in surface charge density suggest a spatial separation for these four membrane components.     

Gallamine triethiodide selectively blocks voltage-gated potassium channels in Ranvier nodes

The effects of gallamine on ionic currents in single intact Ranvier nodes of the toad Xenopus were investigated. The following fully reversible effects were observed: 1. With a test concentration of 1 mmol/l the current-voltage relation of steady-state potassium currents, IK ss exhibited a complete block of IK ss up to about V = 110 mV; with stronger depolarisations the block was incomplete. The peak sodium currents, in contrast, were not affected. 2. At the same test concentration the potassium permeability constant PK was reduced by 92% from its normal value, while the sodium permeability constant PNa decreased by only 8%. 3. Concentration-response relations of the block of PK yielded an apparent dissociation constant of 30 micromol/l and a steepness parameter of unity. Patch-clamp experiments on cloned Kv1.1, Kv1.2, Kv1.3 and Kv3.1 channels yielded apparent dissociation constants of 86, 19, >100 and 121 micromol/l, respectively. Our findings show that gallamine is particularly well suited for separating potassium and sodium currents in axonal current ensembles. They also strongly suggest that potassium currents in Ranvier nodes of Xenopus are mainly carried by an ensemble of Kv1.1 and 1.2 channels.     

The Ventral Photoreceptor Cells of Limulus : III. A voltage-clamp study

In the dark, the ventral photoreceptor of Limulus exhibits time-variant currents under voltage-clamp conditions; that is, if the membrane potential of the cell is clamped to a depolarized value there is an initial large outward current which slowly declines to a steady level. The current-voltage relation of the cell in the dark is nonlinear. The only ion tested which has any effect on the current-voltage relation is potassium; high potassium shifts the reversal potential towards zero and introduces a negative slope-conductance region. When the cell is illuminated under voltage-clamp conditions, an additional current, the light-induced current, flows across the cell membrane. The time course of this current mimics the time course of the light response (receptor potential) in the unclamped cell; namely, an initial transient phase is followed by a steady-state phase. The amplitude of the peak transient current can be as large as 60 times the amplitude of the steady-state current, while in the unclamped cell the amplitude of the peak transient voltage never exceeds 4 times the amplitude of the steady-state voltage. The current-voltage relations of the additional light-induced current obtained for different instants of time are also nonlinear, but differ from the current-voltage relations of the dark current. The ions tested which have the greatest effect on the light-induced current are sodium and calcium; low sodium decreases the current, while low calcium increases the current. The data strongly support the hypothesis that two systems of electric current exist in the membrane. Thus the total ionic current which flows in the membrane is accounted for as the sum of a dark current and a light-induced current.     

Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance

Summary Potassium currents of various durations were obtained from squid giant axons voltage-clamped in artificial seawater solutions containing sufficient tetrodotoxin to block the sodium conductance completely. From instantaneous potassium current-voltage relations, the reversal potentials immediately at the end of these currents were determined. On the basis of these reversal potential measurements, the potassium ion concentration gradient across the membrane was shown to decrease as the potassium current duration increased. The kinetics of this change was shown to vary monotonically with the potassium ion efflux across the membrane estimated from the integral over time of the potassium current divided by the Faraday, and to be independent of both the external sodium ion concentration and the presence or absence of membrane series resistance compensation. It was assumed that during outward potassium current flow, potassium ions accumulated in a periaxonal space bounded by the membrane and an external diffusion barrier. A model system was used to describe this accumulation as a continuous function of the membrane currents. On this basis, the mean periaxonal space thickness and the permeability of the external barrier to K⁺ were found to be 357 Å and 3.21×10^–4 cm/sec, respectively. In hyperosmotic seawater, the value of the space thickness increased significantly even though the potassium currents were not changed significantly. Values of the resistance in series with the membrane were calculated from the values of the permeability of the external barrier and these values were shown to be roughly equivalent to series resistance values determined by current clamp measurements. Membrane potassium ion conductances were determined as a function of time and voltage. When these were determined from data corrected for the potassium current reversal potential changes, larger maximal potassium conductances were obtained than were obtained using a constant reversal potential. In addition, the potassium conductance turn-on with time at a variety of membrane potentials was shown to be slower when potassium conductance values were obtained using a variable reversal potential than when using a constant reversal potential.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings

Summary Electrical currents associated with sodium-coupled alanine transport in mouse pancreatic acinar cells were studied using the method of whole-cell recording with patch pipettes. Single cells or small clusters of (electrically coupled) cells were isolated by collagenase treatment. The composition of the intracellular solution could be controlled by internal perfusion of the patch pipette. In this way both inward and outward currents could be measured under zero-trans conditions, i.e., with finite concentrations of sodium andl-alanine on one side and zero concentrations on the other. Inward andoutward currents for equal but opposite concentration gradients were found to be of similar magnitude, meaning that the cotransporter is functionally nearly symmetric. The dependence of current on the concentrations of sodium andl-alanine exhibited a Michaelis-Menten behavior. From the sodium-concentration dependence of current as well as from the reversal potential of the current in the presence of an alanine-concentration, gradient, a sodium/alanine stoichiometric ratio of 1:1 can be inferred. The finding that N-methylated amino acids may substitute, forl-alanine, as well as the observed pH dependence of currents indicate that the pancreatic alanine transport system is similar to (or identical with) the A-system which is widespread in animal cells. The transport system is tightly coupled with respect to Na⁺; alanine-coupled inward flow of Na⁺ is at least 30 times higher than uncoupled Na⁺ flow mediated by the cotransporter. The current-voltage characteristic of the cotransporter could be (approximately) determined from the difference of transmembrane current in the presence and in the absence ofl-alanine. The sodium-concentration dependence of the current-voltage characteristic indicates that a Na⁺ ion approaching the binding site from the extracellular medium has to cross part of the transmembrane electric field.     

Membrane and junctional properties of dissociated frog lens epithelial cells

Summary Individual cells and cell pairs were isolated from frog lens epithelium. Individual cells were whole cell voltage clamped and the current-voltage relationship was determined. The cells had a mean resting voltage of –54.3 mV and a mean input resistance of 1.4 G. The current-voltage relationship was linear near the cell resting voltage, but showed decreased resistance with large depolarization or hyperpolarization. Junctional currents between pairs of cells were recorded using the dual whole cell voltage-clamp technique. The corrected junctional resistance was 15.5 M (64.5 nS). The junctional current-voltage relationship was linear. A combination of ATP and cAMP, in the electodes, stabilized junctional resistance. Currents recorded when uncoupling was nearly complete, showed evidence of single connexon gating events. A single-channel conductance of about 100 pS was prominent. Dye spread between isolated cell pairs was demonstrated using Lucifer Yellow CH in a whole cell configuration. Photodamage to the cells due to the dye was apparent. Dye loaded cells, in the presence of exciting light, showed decreased resting voltages, decreased input resistances and morphological changes. Glutathione (20mm) delayed this damage.     

Characteristics of sodium tail currents in Myxicola axons. Comparison with membrane asymmetry currents.

Sodium currents after repolarization to more negative potentials after initial activation were digitally recorded in voltage-clamped Myxicola axons compensated for series resistance. The results are inconsistent with a Hodgkin-Huxley-type kinetic scheme. At potentials more negative than -50 mV, the Na+ tails show two distinct time constants, while at more positive potentials only a single exponential process can be resolved. The time-course of the tail currents was totally unaffected when tetrodotoxin (TTX) was added to reduce gNa to low values, demonstrating the absence of any artifact dependent on membrane current. Tail currents were altered by [Ca++] in a manner consistent with a simple alteration in surface potential. Asymmetry current "off" responses are well described by a single exponential. The time constant for this response averaged 2.3 times larger than that for the rapid component of the Na+ repolarization current and was not sensitive to pulse amplitude or duration, although it did vary with holding potential. Other asymmetry current observations confirm previous reports on Myxicola.     

Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

Summary The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 m) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to +80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

An improved vaseline gap voltage clamp for skeletal muscle fibers

A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the "disk" equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5 degrees C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with sodium permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and sodium currents. The peak inward sodium current density at 5 degrees C is 3.7 mA/cm2. At 5 degrees C the sodium currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular system. Comparison of recorded currents with simulations shows that while the transverse tubular system has regenerative sodium currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage clamp control.     

Use of current-voltage diagrams in locating peak energy barriers in cell membranes

Summary The current-voltage relations obtained by integrating the Nernst-Planck equations for a variety of energy profiles are obtained. A simple and approximate method for comparing these relations is described. The method is based on using a linearized transform of current-voltage relations for an Eyring single barrier model. A parameter, , related to the location of the single barrier in the Eyring model, and to the shape of the barrier in other models, is readily obtained from the slopes of the linearized relations. It is then a simple matter to determine whether a given current-voltage relation allows discrimination between any particular energy profiles. The results show that the equivalent Eyring model does not always place the peak energy barrier in the same position as other models and that quite large errors in the assignment of position may be made if such a model is used. The results are also used to test the ability of some experimental current-voltage diagrams to discriminate between various energy profiles.     

Inactivation of the Sodium Current in Myxicola Giant Axons : Evidence for coupling to the activation process

Experiments were conducted on Myxicola giant axons to determine if the sodium activation and inactivation processes are coupled or independent. The main experimental approach was to examine the effects of changing test pulses on steady-state inactivation curves. Arguments were presented to show that in the presence of a residual uncompensated series resistance the interpretation of the results depends critically on the manner of conducting the experiment. Analytical and numerical calculations were presented to show that as long as test pulses are confined to an approximately linear negative conductance region of the sodium current-voltage characteristic, unambiguous interpretations can be made. When examined in the manner of Hodgkin and Huxley, inactivation in Myxicola is quantitatively similar to that described by the h variable in squid axons. However, when test pulses were increased along the linear negative region of the sodium current-voltage characteristic, steady-state inactivation curves translate to the right along the voltage axis. The shift in the inactivation curve is a linear function of the ratio of the sodium, conductance of the test pulses, showing a 5.8 mv shift for a twofold increase in conductance. An independent line of evidence indicated that the early rate of development of inactivation is a function of the rise of the sodium conductance.     

Agonistic effects of xanthines on tetrodotoxin-sensitive sodium channels in the rat dorsal root ganglion neurons

The effects of four xanthine derivatives, caffeine, caffeine benzoate, theophylline, and bromtheophylline, on sodium channels in internally perfused rat dorsal root ganglion neurons were studied under voltage-clamp and whole-cell patch-clamp conditions. Reversible acceleration, enhancement of the amplitude of sodium currents, and shifts of the current-voltage relation (plotted for their maxima), as well as of the steady-state inactivation curve toward more negative potentials, were observed at external applications of the above substances in the concentrations of 0.2–4.0 mM. Under long exposures, inactivation of sodium currents became slower in a part of the cells. Yet, when the exposure to 4 mM or higher concentrations was longer than 10 min, a rise in the passive conductance was obvious, and functional state of the cells became worse. Blocking effects of the xanthine derivatives on transient or delayed potassium currents were not observed. Thus, agonistic action of xanthines on sodium channels has been demonstrated, and it is supposed that a considerable component of their pharmacological effects is provided by the action on Na⁺/Ca²⁺ exchange.     

Acetylcholine-induced current in perfused rat myoballs

Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current- voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface.     

Voltage clamp of the cardiac sodium current at 37 degrees C in physiologic solutions.

The cardiac sodium current was studied in guinea pig ventricular myocytes using the cell-attached patch voltage clamp at 37 degrees C in the presence of 145 mM external sodium concentration. When using large patch pipettes (access resistance, 1-2 M omega), the capacity current transient duration was typically 70 microseconds for voltage clamp steps up to 150 mV. At 37 degrees C the maximum inward sodium current peaked in approximately 200 microseconds after the onset of a clamp step and at this strong depolarization, less than 10% of the sodium current developed during the capacity transient. The sodium current developed smoothly and the descending limb of the current-voltage relationship usually spanned a range of 40 mV. Moreover, currents reduced by inactivation of sodium channels could be scaled to superimpose on the maximum current. Current tails elicited by deactivation followed a monoexponential time course that was very similar for currents of different sizes. Data obtained over a range of temperatures (15 degrees-35 degrees C) showed that the steady-state inactivation and conductance-voltage curves were shifted to more negative voltages at lower temperatures. These results demonstrate the feasibility of investigating the sodium current of mammalian cardiac cells at 37 degrees C in normal physiological solutions.     

Discontinuous feedback amplifier: principles and application to the measurement of transmembrane potentials and currents of frog atrial fibers

The study and achievement of a discontinuous feedback amplifier to measure membrane potentials and currents in frog atrial fibres using the double sucrose gap technique was achieved. It was shown that, with the present device, the effects of the resistance in series with the membrane resistance and the membrane capacity on the measure of cardiac membrane potentials and fast currents are markedly reduced.     

A Theoretical Study on the Sucrose Gap Technique as Applied to Multicellular Muscle Preparations: III. Methodical Errors in the Determination of Inward Currents

The analysis of errors associated with saline-sucrose interdiffusion in sucrose gap experiments on multicellular muscle preparations described in two previous papers (Lammel, E., 1981, Biophys. J., 36:533-553, 555-573) is extended to the determination of current-voltage relations that contain an activated inward current component. The membrane current-voltage (i_t-V_m) relation used in the computations was N-shaped and consisted of two components, an outward (background) current (i_bg) with properties of anomalous (inward-going) membrane rectification, and an inward current (i_s) resembling the slow inward current of cardiac muscle. Reconstruction of current-voltage relations, which simulate those determined experimentally, indicates that in the potential range in which the total membrane current (i_t) is outward, i_t is measured too high, whereas it is measured too low in the range of net inward current. Reversal potentials of the inward and outward components are both shifted to more negative values, that of the inward current being more affected. Simulation of the experimental approach to evaluate i_s as the difference between i_t and i_bg shows that errors that produce values too high for i_bg are partly compensated by errors that lead to values of the net inward component that are too low. The basic features of the distorting effects analyzed are independent of different assumptions made on the selectivity of the slow inward current channels. They are related to currents emerging from the sucrose compartment (local circuit as well as externally applied currents).