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Biomedical subjects

F Bezanilla

Publications and source records attributed to F Bezanilla.

At least 91 records · Page 5Linked to original sources
PubMed1983

Sodium and gating current time shifts resulting from changes in initial conditions.

The sodium and gating currents of the squid giant axon elicited by a depolarizing pulse are delayed, with little change in shape, as a result of a hyperpolarizing prepulse. The delays are almost completely saturated, at approximately 45 microseconds, for prepulses to -140 mV. At 8 degrees C they develop with time constants of between 60 and 180 microseconds for prepulses in the -130- to -150-mV range. There is a correlation between the extra charge moved during the gating current and the increase in the time delay of the sodium current as the magnitude of the hyperpolarizing prepulse is increased. These results strengthen the conclusion that the gating current is indeed closely associated with the process of sodium channel opening and provide information concerning the kinetics of the early steps, which are hidden in ionic current measurements. The main features of the gating and sodium current time shifts and the correlation between charge movement and time shifts are duplicated by a sequential six-state model for sodium activation.

Animals↗
PubMed1983

Induced capacitance in the squid giant axon. Lipophilic ion displacement currents.

Voltage-clamped squid giant axons, perfused internally and externally with solutions containing 10(-5) M dipicrylamine (DpA-), show very large polarization currents (greater than or equal to 1 mA/cm2) in response to voltage steps. The induced polarization currents are shown in the frequency domain as a very large voltage-and frequency-dependent capacitance that can be fit by single Debye-type relaxations. In the time domain, the decay phase of the induced currents can be fit by single exponentials. The induced polarization currents can also be observed in the presence of large sodium and potassium currents. The presence of the DpA- molecules does not affect the resting potential of the axons, but the action potentials appear graded, with a much-reduced rate of rise. The data in the time domain as well as the frequency domain can be explained by a single-barrier model where the DpA- molecules translocate for an equivalent fraction of the electric field of 0.63, and the forward and backward rate constants are equal at -15 mV. When the induced polarization currents described here are added to the total ionic current expression given by Hodgkin and Huxley (1952), numerical solutions of the membrane action potential reproduce qualitatively our experimental data. Numerical solutions of the propagated action potential predict that large changes in the speed of conduction are possible when polarization currents are induced in the axonal membrane. We speculate that either naturally occurring substances or drugs could alter the cable properties of cells in a similar manner.

Animals↗
PubMed1983

An optical determination of the series resistance in Loligo.

The resistance in series with the membrane capacitance in the giant axon of the squid Loligo pealei was measured using potentiometric probes that exhibit absorbance changes proportional to the voltage across the plasma membrane proper. The method relies upon the fact that a voltage drop across the series resistance produces a deviation in the true transmembrane voltage from that imposed by a voltage clamp. Optical measurement of the true transmembrane potential, together with electrical measurement of the ionic current, permits the immediate determination of the series resistance by Ohm's law. An alternative method monitored the amount of electronic series resistance compensation required to force the optical signal to match the shape of the reference potential. The value of the series resistance measured in artificial seawater was 3.78 +/- 0.95 omega X cm2. The estimated value of the contribution of the Schwann cell layer to the series resistance was 2.57 +/- 0.89 omega X cm2.

Animals↗
PubMed1982

Distribution and kinetics of membrane dielectric polarization. 1. Long-term inactivation of gating currents.

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.

Animals↗
PubMed1982

Distribution and kinetics of membrane dielectric polarization. II. Frequency domain studies of gating currents.

We have studied the admittance of the membrane of squid giant axon under voltage clamp in the absence of ionic conductances in the range of 0-12 kHz for membrane potentials (V) between --130 and 70 mV. The admittance was measured at various holding potentials (HP) or 155 ms after pulsing from a given holding potential. Standard P/4 procedure was used to study gating currents in the same axons. We found that the membrane capacity Cm (omega) is voltage as well as frequency dependent. For any given V, the voltage-dependent part of the membrane capacitance has a maximum as the frequency approaches zero and requires at least a two-time constant equivalent circuit to be described. When the holding potential is varied, the voltage-dependent capacitance follows a bell-shaped curve with a maximum change of 0.15 muF/cm2 at about --60 mV. With the pulse method, the maximum is at --40 mV for HP = --70 and it shifts to --70 mV for HP = 0. The shift in the maximum of the voltage-dependent capacitance is consistent with the shift in the charge (Q) vs. V curve observed in our experiments with regular P/4 procedure when the HP is varied. Our data can be explained qualitatively by a four-state model for the sodium channel gating, where a charged particle can move within the field and interact with another particle not affected by the field.

Animals↗
PubMed1980

Diffusion models for the squid axon Schwann cell layer.

The Schwann cell, basement membrane, and connective tissue layers that surround the squid giant axon and constitute barriers to diffusion, were modeled in a number of ways to analyze various experimental results. The experiments considered are (a) the time-course of the potassium concentration in the space between the Schwann cell and the axon membrane (from now on referred to as the F-H space) after an initial loading, (b) the time-course of sodium concentration in the F-H space after a sudden change in the sodium concentration in the external fluid; (c) the time-course of the concentration of tetrodotoxin (TTX) or saxitoxin (STX) in the F-H space after a sudden change in external concentration, including (or not) the effects of specific binding of TTX or STX to sites on the axon membrane and nonsaturable binding to sites in the F-H space or in the spaces (clefts) between Schwann cells; (d) the effects of the F-H space, clefts, and diffusion into the clefts from the outside (from now on referred to as convergence into the clefts) on the measured series resistance.The analysis shows that (1) in no case is it necessary to include the effects of the convergence into the clefts from the outside; (2) in case a, the basement membrane, connective tissue layers, and the unstirred layer may be neglected, i.e., the clefts are rate limiting; (3) in case b the clefts may be neglected, i.e., the unstirred layer is rate limiting; (4) in most cases the clefts may be replaced by an equivalent thin diffusion barrier.

Animals↗
PubMed1980

Current recorded from a cut-open giant axon under voltage clamp.

Squid giant axons were cut open and the resulting membrane sheet was positioned in a chamber separating two compartments. Under voltage clamp, normal sodium, potassium, and gating currents could be recorded for several hours. This preparation allows direct access to the internal side of the axonal membrane and should prove useful for recording of ionic currents, gating currents, and channel-induced current fluctuations in the same membrane sheet.

Animals↗
PubMed1978

Temperature effects on gating currents in the squid giant axon.

The effects of temperature (3 degrees-26 degrees C) on the nonlinear components of the displacement current were measured in internally perfused, voltage clamped squid axons. Steps of potential were applied from a holding potential of -70mV (outside ground) to values from -130 to +70mV and either the current or its integral (charge) was recorded as a function of time. For that component of the charge movement not linearly related to voltage, the total charge moved in a few milliseconds (about 1,500 electronic charges/micron2) between saturation limits (e.g. -100mV to +50mV) showed an apparent increase of 13 +/- 5% for a 10 degrees C rise in temperature. Attempts to fit the falling phase of the gating current (or charge) with the sum of two exponentials showed temperature effects on both components but there was considerable scattering. At short times, records for current or charge made at 16 degrees C, expanded by a factor alpha, superimposed on those made at 6 degrees C for alpha about 1.6. For long times alpha was about 2.3.

Axons↗
PubMed1978

Nile blue fluorescence signals from cut single muscle fibers under voltage or current clamp conditions.

A method is presented for recording extrinsic optical signals from segments of single skeletal muscle fibers under current or voltage clamp conditions. Such segments, which are cut from intact fibers, are maintained in a relaxed state, while exhbiting otherwise normal physiological properties, including healthy delayed rectifier currents. Extrinsic fluorescence changes are demonstrated, using the permeant potentiometric probe, Nile Blue A. These changes vary nonlinearly with the controlled surface membrane potential, in a manner which suggests that they arise from potential changes in the sarcoplasmic reticulum. According to this interpretation, a simple model based on the gating charge movement implicated in excitation-contraction coupling, provides a self-consistent description of the voltage dependence of the signal that requires no additional parameters.

Action Potentials↗
PubMed1977

Inactivation of the sodium channel. I. Sodium current experiments.

Inactivation of sodium conductance has been studied in squid axons with voltage clamp techniques and with the enzyme pronase which selectively destroys inactivation. Comparison of the sodium current before and after pronase treatment shows a lag of several hundred microseconds in the onset of inactivation after depolarization. This lag can of several hundred microseconds in the onset of inactivation after polarization. This lag can also be demonstrated with double-pulse experiments. When the membrane potential is hyperpolarized to -140 mV before depolarization, both activation and inactivation are delayed. These findings suggest that inactivation occurs only after activation are delayed. These findings suggest that inactivation occurs only after activation; i.e. that the channels must open before they can inactivate. The time constant of inactivation measured with two pulses (tau(c)) is the same as the one measured from the decay of the sodium current during a single pulse (tau(h)). For large depolarizations, steady-state inactivation becomes more incomplete as voltage increases; but it is relatively complete and appears independent of voltage when determined with a two- pulse method. This result confirms the existence of a second open state for Na channels, as proposed by Chandler and Meves (1970. J. Physiol. [Lond.]. 211:653-678). The time constant of recovery from inactivation is voltage dependent and decreases as the membrane potential is made more negative. A model for Na channels is presented which has voltage-dependent transitions between the closed and open states, and a voltage-independent transition between the open and the inactivated state. In this model the voltage dependence of inactivation is a consequence of coupling to the activation process.

Action Potentials↗
PubMed1977

Inactivation of the sodium channel. II. Gating current experiments.

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.

Action Potentials↗
PubMed1977

A low-cost signal averager and data-acquisition device.

A low-cost data-acquisition device, which can be used as a signal averager or temporary data buffer, is described. It consists of 1) an input stage built with a multiplexer, sample-and-hold amplifier, and an analog-to-digital converter; 2) a memory section made of static shift registers with recirculate capability; 3) an output stage built with a demultiplexer and a digital-to-analog converter and, 4) a control module which provides the logic signals to operate the machine. Basic diagrams are presented and several applications are described.

Electronics↗