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E Rojas

Publications and source records attributed to E Rojas.

At least 235 records · Page 13Linked to original sources
PubMed1975

The rate of action of tetrodotoxin on sodium conductance in the squid giant axon.

When tetrodotoxin is applied to or washed away from the squid giant axon, the rates at which the sodium conductatnce is blocked and unblocked are an order of magnitude smaller than those reported for the isolated node of Ranvier. This slowing is to be expected if in squid the tetrodotoxin binding sites act as a saturable sink in series with the barrier to free diffusion imposed by the presence of the Schwann cell. A comparison has been made between the rates observed experimentally and those calculated for a computer model of the system, in order to estimate the apparent density in the membrane of both specific and non-specific tetrodotoxin binding sites. The figure thus obtained for the number of sodium channels in the squid giant axon, several hundred per square micrometre, agrees well with those derived from other lines of argument.

Animals↗
PubMed1975

On the relation between displacement currents and activation of the sodium conductance in the squid giant axon.

The early time course of the current passing across the membrane in squid giant axons in which the ionic currents have been blocked reveals substantial asymmetries during and after the application of hyperpolarizing and depolarizing voltage-clamp pulses of identical size. Since the integral of the 'on' and 'off' current transients is zero, these currents must result from charge movements confined to the membrane and, therefore, they are nonlinear displacement currents. The steady state rearrangement of the charges as a consequence of sudden displacements of the membrane potential is consistent with a Boltzmann distribution of charges between two states characterized by different energy levels. Following changes in membrane potential the charges undergo a first order transition between these states. The relaxation time constant for the transition at a given temperature is a function of membrane potential. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.

Action Potentials↗
PubMed1975

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).

Animals↗
PubMed1975

Displacement currents in the node of Ranvier. Voltage and time dependence.

The time course of the membrane currents in the node of Ranvier in which the sodium and potassium conductances have been blocked reveals asymmetries during and after the application of depolarizing and hyperpolarizing voltage-clamp pulses of identical size. Since 1. the integrals of the "on" and "off" current transients were found to be equal and opposite, 2. the change displaced reached saturation (about 140-10-minus 15 C/node) when the internal potential was taken to a sufficiently positive value during the depolarizing pulses and, 3. the size of the charge transferred was unaffected by temperature although its time constant had a large temperature coefficient (Q10 = 2.4), these currents to our opinion must result form charge movements confined to the membrane and, therefore, can be considered as non-liner displacement currents. The steady-state rearrangement of the charges is consistent with a Boltzmann distribution of charges (effective valence z = 1.65) between two configurations characterized by different energy levels. The midpoint potential of the distribution curve is -33.7mV and its maximum slope, kT/ZE, is 14.9 mV. Following changes in membrane potential the charges undergo a first order transition between these states. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.

Action Potentials↗
PubMed1975

Simultaneous measurements of magnesium, calcium and sodium influxes in perfused squid giant axons under membrane potential control.

1. Giant axons from the squids Dosidicus gigas, Loligo forbesi and Loligo vulgaris were internally perfused with 550 or 275 mM KF plus sucrose and bathed in artificial sea water containing 45Ca, 28Mg or mixtures of 45Ca-28Mg or 45Ca-22Na. Resting influxes and extra influxes during voltage-clamp pulses were measured by collecting and counting the internal perfusate. 2. For Dosidicus axons in 10 mM-CaCl2 the resting influx of calcium was 0-016 +/- 0-007 p-mole/cm2 sec and a linear function of external concentration. For two experiments in 10 and 84-7 mM-CaCl2, 100 nM tetrodotoxin had no effect. Resting calcium influx in 10 mM-CaCl2 was 0-017 +/- 0-013 p-mole/cm2 sec for Loligo axons. 3. With 55 mM-MgCl2 outside the average resting magnesium influx was 0-124 +/- 0-080 p-mole/cm2 sec for Loligo axons. Discarding one aberrant point the value is 0-105 +/- 0-046 which is not significantly different from the resting calcium influx for Dosidicus fibres in 55 mM-CaCl2, given as 0-094 p-mole/cm2 sec by the regression line shown in Fig. 1. In two experiments 150 nM tetrodotoxin had no effect. 4. With 430 mM-NaCl outside 100 nM tetrodotoxin reduced the average resting influx of sodium in Dosidicus axon from 27-7 +/- 4-5 to 25-1 +/- 6-2 p-mole/cm2 sec and for Loligo fibres in 460 mM-NaCl from 50-5 +/- 4 to 20 +/- 8 p-mole/cm2 sec. 5. Using depolarizing pulses of various durations, the extra calcium influx occurred in two phases. The early phase was eliminated by external application of tetrodotoxin. The results of analysis are consistent with, but do not rigorously demonstrate, the conclusion that the tetrodotoxin sensitive calcium entry is flowing through the normal sodium channels (cf. Baker, Hodgkin & Ridgway, 1971). 6. Measurements of extra influxes using 22Na and 45Ca simultaneously indicate that the time courses of tetrodotoxin sensitive calcium and sodium entry are similar but not necessarily identical. It is very doubtful that any significant calcium entry occurs before the sodium or is involved in the activation of the sodium system. 7. These measurements confirm for Loligo, as previously shown for Dosidicus axons, that the magnitude and time course of the sodium entry during a depolarizing pulse deduced from electrical measurements is the same as that measured with 22Na. 8. Using 28Mg, or mixtures of 45Ca and 28Mg, we observed a single phase of magnesium entry which was insensitive to external tetrodotoxin or internal tetraethyl ammonium. The magnitude of the magnesium influx was considerably greater than the calcium extra entry and large enough to have been detected in the experiments of Meves & Vogel (1973) if it represented current. 9. We suggest the possibility that the calcium and magnesium extra influxes, after external treatment with tetrodotoxin, during a depolarizing pulse, do not contribute to the measured current.

Animals↗
PubMed1974

Kinetics and steady-state properties of the charged system controlling sodium conductance in the squid giant axon.

1. Asymmetries in the early time course of the displacement current passing across the membrane after application of equal voltage-clamp pulses in the two directions have been investigated in the squid giant axon. Before making the measurements, Na current was blocked by removal of external Na and treatment with tetrodotoxin. Potassium current was usually blocked by perfusion with CsF, but some experiments were done with intact axons. A signal averaging technique was used to eliminate the symmetrical components of the membrane current.2. The asymmetrical current had a contribution of appreciable size attributed to the movement of mobile charges or dipoles in the membrane. This was manifested as an outward current rising rapidly to a peak on depolarization of the membrane and then declining exponentially to zero, followed at the end of the pulse by an inward surge of current with a similar time course. There was also a sustained flow of current outwards during the pulse, arising from ionic leakage with a rectifying characteristic.3. The identification of the exponentially changing current component with the displacement of charged particles forming an integral part of the membrane was supported by the demonstration that the total transfer of charge was equal and opposite at the beginning and end of the pulse, that it reached saturation when the internal potential was taken to a sufficient positive value, and that its size was unaffected by temperature, although its time constant had a large temperature coefficient.4. The disposition of the mobile charges in the steady state was shown to obey a Boltzmann distribution. At the midpoint of the distribution curve, the proportion of the charge displaced underwent an e-fold change for a 19 mV change in potential. The effective valency of the particles, that is their actual charge multiplied by the fraction of the electric field acting on them, was therefore 1.3.5. The total quantity of mobile charge was estimated as about 1500 x 10(-12) C for 0.05 cm(2) of membrane, corresponding to some 1900 charges/mum(2).6. The identification of these mobile charges with the gating particles responsible for controlling Na conductance was supported by the findings that (a) their time constants were the same as those of Hodgkin & Huxley's ;m' system, both in absolute magnitude and in their dependence on potential and temperature, (b) the transition potential at which the charges were evenly distributed on the two sides of the membrane also agreed with that for the ;m' system in intact axons, and its value was similarly shifted in a positive direction by a reduction in internal ionic strength or by raising the external Ca concentration, (c) comparison of the steepness of the curves governing on the one hand the steady-state distribution of the mobile charges and on the other the Na conductance, suggested that an effective cooperation of the charges in groups of three was involved, again in excellent agreement with the ;m' system.7. Displacement of the mobile charges was unaffected by external pH over the range 5-8, but preliminary observations showed that 1% procaine reduced the total charge transfer to somewhat less than 40% of the initial value, and roughly halved the time constant.

Animals↗
PubMed1974

Calcium influxes and tension development in perfused single barnacle muscle fibres under membrane potential control.

1. Single giant barnacle muscle fibres from Megabalanus psittacus (Darwin) were used to measure the Ca entry and the development of tension in the fibres under membrane potential control.2. Fibres bathing in 60 mm-MgCl(2) sea water, free of Ca, did not develop tension with sudden displacements of the membrane potential towards more positive values. This failure to develop tension with depolarizations was observed with and without the internal application of Ca buffers.3. Fibres bathing in artificial sea water with either 10, 20, 60 or 100 mm-CaCl(2) developed tension with depolarization even after 60 min of internal perfusion of the fibres with solution containing no Ca buffers. In this case the maximum tension recorded during a voltage clamp run decreased with time from nearly 2.5 to 0.2 kg/cm(2). However, addition of 10 mm-Tris-EGTA (ethyleneglycol-bis (beta-aminoethyl ether) N, N' - tetraacetic acid) to the perfusing solution rapidly eliminated the development of tension; after 10 min of internal perfusion with Ca buffers no tension could be elicited by electrical stimulation.4. Ca-influx determinations were carried out only in the fibres in which the outward K(+) currents were blocked by internal application of TEA (tetra-ethylammonium). The ratio of ;measured extra Ca influx/computed ionic flux of divalent cations during the inward current' was 1.06 +/- 0.41.5. For fibres bathed in either natural sea water or in artificial sea water with various concentrations of Ca, the temporal course of development of isometric tension was similar to the temporal course of the integral of the inward current due to Ca(2+).6. In a fibre from M. psittacus bathing in natural sea water the calculated extra entry of Ca required to increase its internal concentration to about 50 mum was 500 p-mole per depolarization (60 mV); while the corresponding average influx calculated from the inward current record in natural sea water is 474 p-mole.7. Evidence was obtained for the accumulation of Ca in an internal compartment.

Acetates↗
PubMed1973

Destruction of sodium conductance inactivation in squid axons perfused with pronase.

We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g(Na)). At the level of a single channel, Pronase probably acts in an all-or-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces g(Na), possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.

Action Potentials↗
PubMed1973

Calcium and potassium systems of a giant barnacle muscle fibre under membrane potential control.

1. Single barnacle muscle fibres from Megabalanus psittacus (Darwin) were internally perfused and the effects of various internal and external solutions on voltage clamp currents were examined.2. The usual internal solution was 180 mM-K(+) aspartate (osmotic pressure adjusted to 1000 m-osmole by adding sucrose). Fibres perfused with this solution gave an average resting potential of -55 +/- 5 mV (all potentials are referred to the external solutions as ground). Further increase in internal K concentration depolarized the fibres.3. With membrane current control the total capacitance, referred to apparent membrane surface area, was 21.2 +/- 2.4 muF/cm(2).4. Under normal conditions, with demonstrably good longitudinal space clamp control, voltage clamp currents associated with certain depolarizing pulses showed oscillations. These oscillations were reduced in frequency and magnitude by lowering the temperature from 20 to 10 degrees C, by eliminating the inward currents with external Ca-free saline or by reducing the outward currents with internal tetraethylammonium (TEA) or replacement of internal K by Cs.5. With a Na- and Ca-free, 60 mM-MgCl(2) solution outside depolarizing voltage clamp pulses produced only outward currents. On repolarization the current tail reversed direction at about -70 mV for pulses of less than 50 msec duration. For longer pulses this reversal potential was less negative, suggesting an accumulation of external, or depletion of internal K.6. Both the size of the outward currents and the rate at which they reached their maximum value increased with temperature. The activation energy for the rate constant was about 63 kJ/mole.7. Fibres bathed in Na- and Mg-free, 60 mM-CaCl(2) saline were excitable. After replacement of the internal K(+) with Cs(+) or adding 60 mM-TEA to the internal solution only sustained inward currents were recorded with depolarization.8. Sustained inward currents could be reduced by external application of 5 mM-LaCl(3). Tetrodotoxin was not effective even at a concentration of 1000 nM.9. The rate at which these inward currents reached a maximum value increased with increase in temperature of the bathing solution with an activation energy of the order of 42 kJ/mole.10. The reversal potential of the inward currents changed with the level of internal Ca ions. For a fibre perfused without ethyleneglycol-bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) this reversal potential was 175 mV (internal free Ca 5 x 10(-7)M), and was 196 mV for a fibre perfused with 20 mM-Tris EGTA (internal free Ca 0.26 x 10(-8)M).11. We propose an electrical equivalent circuit to account for most of the observed electrical properties of barnacle muscle fibres. In this model the Ca and the K system are located at different anatomical places and they interact through a series resistance.

Animals↗
PubMed1973

Effects of anions and cations on the resting membrane potential of internally perfused barnacle muscle fibres.

1. Single barnacle muscle fibres from Megabalanus psittacus (Darwin) were internally perfused with a number of K salt solutions (200 mM) which were made isotonic to the barnacle saline with sucrose.2. 200 mM-K acetate solution, in general, was found to be more effective than other solutions of K salts in generating and maintaining stable resting membrane potential of -56.0 +/- 0.7 mV (all potentials are referred to the external solutions as ground). The various K salts, on the basis of the magnitude of the resting potential they generated in the muscle fibres, followed the sequence, acetate > isethionate > aspartate > glutamate > fluoride > monohydrogen phosphate > succinate > citrate > sulphate > oxalate > iodobenzoate > ferrocyanide > chlorate > nitrate > chloride > thiocyanate > iodide > bromide > cyanide.3. The resting potential in muscle fibres perfused with solutions of acetate, aspartate and glutamate increased linearly with the logarithm of the K concentration (slope = 30.4 mV for K acetate and 27.4 for K aspartate and glutamate) when the ionic strength of the solutions was progressively increased from 50 to 650 mM. On the other hand, similar increase of ionic strength beyond 200 mM of solutions of K isethionate, fluoride, monohydrogen phosphate, succinate and citrate depolarized the muscle fibres.4. Perfusion of acetate solutions of other alkali metal ions gave low values for the resting potential and followed the sequence K > Na > Rb > Li > Cs. Also NH(4) and Tris ions gave low values for the resting potential which underwent oscillations associated with the twitching of the fibre and occasionally became positive in value (action potential).5. Addition of tetraethyl ammonium chloride (TEA-Cl), 20-100 mM, to K acetate solutions (200 mM) depolarized the fibre membrane and the consequent reduction of resting potential varied linearly with the logarithm of TEA concentration.6. Replacement of chloride ion by acetate or isethionate in the external solution did not change significantly the resting potential although the values were consistently lower by about 2 mV.7. Complete elimination of K in the external solution and reduction of its ionic strength using sucrose depolarized the muscle fibres by about 27 mV when Na was changed from 475 to 1 mM. Under these conditions, external solutions completely in acetate form gave resting potentials which were more positive than those observed in completely chloride solutions by 6-8 mV.8. Replacement of Na by Li, Tris, choline, tetramethyl or tetraethyl ammonium ion in the external solution made the values of the resting potential more positive (depolarization). Similarly increasing the concentration of K (or Cs or Rb in place of K) by correspondingly decreasing the concentration of Na in the outside solution depolarized the fibres and the resting potential became zero at a concentration of 280 mM (or 308 or 1500 mM for Rb or Cs, respectively) on extrapolation.

Acetates↗