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Biomedical subjects

F Bezanilla

Publications and source records attributed to F Bezanilla.

At least 109 records · Page 6Linked 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

Kinetic properties and inactivation of the gating currents of sodium channels in squid axon.

Associated with the opening and closing of the sodium channels of nerve membrane is a small component of capacitative current, the gating current. After termination of a depolarizing step the gating current and sodium current decay with similar time courses. Both currents decay more rapidly at relatively negative membrane voltages than at positive ones. The gating current that flows during a depolarizing step is diminished by a pre-pulse that inactivates the sodium permeability. A pre-pulse has no effect after inactivation has been destroyed by internal perfusion with the proteolytic enzyme pronase. Gating charge (considered as positive charge) moves outward during a positive voltage step, with voltage dependent kinetics. The time constant of the outward gating current is a maximum at about minus 10 mV, and has a smaller value at voltages either more positive or negative than this value.

Action Potentials↗
PubMed1975

Fluorescence intensity changes associated with contractile activation in frog muscle stained with Nile Blue A.

1. Extrinsic fluorescence intensity changes were studied in frog semitendinosus muscles stained with Nile Blue A in response to electrical stimulation. Muscles were stretched and put into hypertonic solutions to prevent movement. The muscles were illuminated at 90 degrees to their long axis with a narrow beam of light at a central wave-length of 6250 . Fluorescence emission was measured at 90 degrees to the exciting light using a filter which absorbed light of wave-lengths shorter than 6400 . 2. In response to a single stimulus the fluorescence intensity increases briefly. The fluorescence response is propagated at a constant velocity of about 1.5 m/sec. The average ratio of the maximum fluorescence intensity change to the resting fluorescence is 4.5 times 10-3 for supramaximal shocks. The fluorescence intensity change starts early in the falling phase of the action potential. 3. The fluorescence intensity change increases when nitrate replaces chloride and decreases when D2O replaces H2O. The rates of rise and fall of the fluorescence change was unaffected by nitrate replacement of chloride but are slowed where D2O replaces H2O. The rates of rise and fall of the fluorescence change increase with increasing temperature for all solutions used. The peak fluorescence intensity change, however, goes through a maximum at about 17 degrees C for aqueous chloride and nitrate solutions in the range of 10-25 degrees C. With D2O solutions, the peak fluorescence intensity increases monotonically in this range of temperatures. 4. The fluorescence intensity change in response to trains of action potentials are not additive. 5. Depolarization of muscles treated with tetrodotoxin using triangular-shaped fluid electrodes produces an increase in fluorescence at about the same threshold values required to elicit tension in preparations that are not fully stretched. The fluorescence intensity change precedes in time tension development. Near threshold depolarizations, the delay in onset of the fluorescence response can be 80 msec or longer. Byond threshold, delays become shorter and peak responses larger. During maintained depolarization, after the peak response, fluorescence declines to a plateau value. 6. The results suggest that the fluorescence intensity changes are associated with excitation-contraction coupling, possibly with changes in the transmembrane potential of the sarcoplasmic reticulum.

Action Potentials↗
PubMed1974

Gating currents of the sodium channels: three ways to block them.

Preceding the opening of the sodium channels of axon membrane there is a small outward current, gating current, that is probably associated with the molecular rearrangements that open the channels. Gating current is reversibly blocked by three procedures that block the sodium current: (i) internal perfusion with zinc ions, (ii) inactivation of sodium conductance by brief depolarization, and (iii) prolonged depolarization.

Animals↗
PubMed1974

Charge movement associated with the opening and closing of the activation gates of the Na channels.

The sodium current (I(Na)) that develops after step depolarization of a voltage clamped squid axon is preceded by a transient outward current that is closely associated with the opening of the activation gates of the Na pores. This "gating current" is best seen when permeant ions (Na and K) are replaced by relatively impermeant ones, and when the linear portion of capacitative current is eliminated by adding current from positive steps to that from exactly equal negative ones. During opening of the Na pores gating current is outward, and as the pores close there is an inward tail of current that decays with approximately the same time-course as I(Na) recorded in Na-containing medium. Both outward and inward gating current are unaffected by tetrodotoxin (TTX). Gating current is capacitative in origin, the result of relatively slow reorientation of charged or dipolar molecules in a suddenly altered membrane field. Close association with the Na activation process is clear from the time-course of gating current, and from the fact that three procedures that reversibly block I(Na) also block gating current: internal perfusion with Zn(2+), prolonged depolarization of the membrane, and inactivation of I(Na) with a short positive prepulse.

Animals↗
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↗
PubMed1972

Negative conductance caused by entry of sodium and cesium ions into the potassium channels of squid axons.

Internal Cs(+), Na(+), Li(+), and, to a lesser degree, Rb(+) interfere with outward current through the K pores in voltage clamped squid axons. Addition of 100 mM NaF to the perfusion medium cuts outward current for large depolarizations about in half, and causes negative conductance over a range of membrane voltages. For example, suddenly reducing membrane potential from +100 to +60 mv increases the magnitude of the outward current. Internal Cs(+) and, to a small extent, Li(+), also cause negative conductance. Na(+) ions permeate at least 17 times less well through the K pores than K(+), and Cs(+) does not permeate measurably. The results strongly suggest that K pores have a wide and not very selective inner mouth, which accepts K(+), Na(+), Li(+), Cs(+), tetraethylammonium ion (TEA(+)), and other ions. The diameter of the mouth must be at least 8 A, which is the diameter of a TEA(+) ion. K(+) ions in the mouths probably have full hydration shells. The remainder of the pore is postulated to be 2.6-3.0 A in diameter, large enough for K(+) and Rb(+) but too small for Cs(+) and TEA(+). We postulate that Na(+) ions do not enter the narrower part of the pore because they are too small to fit well in the coordination cages provided by the pore as replacements for the water molecules surrounding an ion.

Animals↗
PubMed1972

Sodium dependence of the inward spread of activation in isolated twitch muscle fibres of the frog.

1. The excitatory process travelling along the T-system may be either electrotonic or regenerative. If Na(+) dependent action potential is present in the tubular membranes, high frequency of stimulation might cause a Na(+) depletion in the tubules sufficient to abolish this process.2. We tested this hypothesis by recording tension in isolated muscle fibres stimulated tetanically (up to 60 shocks/sec). In low [Na(+)] solutions, output tension was initially similar to that in normal Ringer, but then fell smoothly to a substantially lower value.3. The activity of individual myofibrils was recorded directly with ciné-micrographs during isotonic contractions while the fibres were stimulated at high frequencies. In low [Na(+)](o) wavy myofibrils appeared in the centre of the fibre and spread towards the periphery, indicating failure of activation. Wavy myofibrils never appeared in normal Ringer.4. Intracellular action potentials recorded during the tetanic stimulation indicated that the inactivated myofibrils present in low [Na(+)] solutions cannot be explained by the changes in size and duration of the action potential.5. Our results strongly suggest the existence of a regenerative Na(+) conductance in the tubular membrane during the inward spread of an excitatory process.

Action Potentials↗
PubMed1970

Nerve membrane excitation without threshold.

Evidence is presented to show that for a squid axon membrane the potential response, V, is a smoothly continuous function of a stimulating current, I. This makes it unlikely that an all-or-none or sharp transition phenomenon is a major factor in the processes by which ions cross the normal squid axon membrane and, probably, other excitable membranes. Spatially uniform V and I were first produced in the squid axon with internal and external electrode arrangements and later by isolating a short length of axon between external pools of sucrose. Under these simplified conditions, direct experiments and calculations based on the Hodgkin-Huxley empirical conductances agree in showing that the maximum response, R, is a continuous, single-valued function of the effect of the stimulus, S. The maximum value of DeltaR/DeltaS decreased steadily as the temperatures were increased from 25 degrees to 38 degrees C. Uncontrolled fluctuations prevented direct observations of DeltaR/DeltaS below 15 degrees C where calculations showed that it rose rapidly as the temperature decreased. Since the conductances are experimental parameters and since DeltaR/DeltaS as calculated from them remained finite and continuous event at 6.3 degrees C, this is experimental evidence against an all-or-none threshold excitation. However there is an all-or-none threshold for the initiation and propagation of an impulse along an axon where V and I are functions of both time and distance.

Animals↗
PubMed1970

Time course of the sodium influx in squid giant axon during a single voltage clamp pulse.

1. Sodium influx measurements were carried out on internally perfused squid giant axons under conditions of membrane potential control.2. The ratio ;measured extra sodium influx/calculated ionic influx from the inward current record' is close to unity and is independent of the duration of the rectangular pulse of membrane potential.3. The variation of the sodium permeability with time during the voltage clamp pulse is obtained by subtracting the sodium influx obtained from the ;tail' current record from the measured extra sodium influx.4. Evidence was obtained indicating that the measured change in sodium permeability during a sudden decrease of the membrane potential from its resting level involves two processes, namely, a transient increase in sodium permeability (activation), and a decrease in sodium permeability (inactivation).5. The extra influx of sodium during a voltage clamp pulse is not decreased by raising the internal sodium concentration from 0 to 100 mM, supporting the validity of the independence principle.

Animals↗
PubMed1970

Sodium and potassium conductance changes during a membrane action potential.

1. A method for turning a membrane potential control system on and off in less than 10 musec is described. This method was used to record membrane currents in perfused giant axons from Dosidicus gigas and Loligo forbesi after turning on the voltage clamp system at various times during the course of a membrane action potential.2. The membrane current measured just after the capacity charging transient was found to have an almost linear relation to the controlled membrane potential.3. The total membrane conductance taken from these current-voltage curves was found to have a time course during the action potential similar to that found by Cole & Curtis (1939).4. The instantaneous current voltage curves were linear enough to make it possible to obtain a good estimate of the individual sodium and potassium channel conductances, either algebraically or by clamping to the sodium, or potassium, reversal potentials. Good general agreement was obtained with the predictions of the Hodgkin-Huxley equations.5. We consider these results to constitute the first direct experimental demonstration of the conductance changes to sodium and potassium during the course of an action potential.

Action Potentials↗
PubMed1970

Time course of the sodium permeability change during a single membrane action potential.

1. A method for measuring the time course of ionic fluxes during a non-propagated membrane action potential is described; the technique combines intracellular perfusion of the squid giant axon with radioactive tracer methods and with a method for controlling the membrane potential during small time intervals.2. The method is used to determine the temporal course of the sodium extra influx during an action potential.3. The results agree with the time course of the permeability change to sodium ions calculated with the Hodgkin & Huxley equations.4. An average extra sodium influx of 7.13 p-mole/cm(2) per action potential was determined at 12 degrees C and of 5.23 p-mole/cm(2) per action potential at 15 degrees C.

Action Potentials↗
PubMed1969

Analysis of the effects of calcium or magnesium on voltage-clamp currents in perfused squid axons bathed in solutions of high potassium.

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15-30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system.

Animals↗