Transport of Na+ inside the giant axon of squid

The transport mechanism of Na ions within the nerve cell was studied by measuring the radioactivity distribution profile of²²Na that had been intracellularly injected into the giant axon. Specifically, we tested whether or not the movement of Na ions is coupled with the process of “fast axonal transport.” Results of our measurements indicate that the intracellular transport of Na⁺ and the fast axonal transport are two independent processes. Very few Na ions are irreversibly sequestered into the axoplasmic vesicles involved in axonal transport. The movement of Na⁺ inside the axon can be modeled by a one-dimension diffusion. The effective diffusion coefficient of the intracellular Na⁺ was determined in this study.     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

Complex admittance of Na+ conduction in squid axon

Summary The complex admittance,Y(p), of squid axon was measured (4-1000 Hz) during step voltage clamp to obtain linear data on Na⁺ conduction.Y(p) is used as a spectroscopic tool to identify Na⁺ and K⁺ conduction, which dominateY(p) at low frequencies and can be separated from each other and from the static capacitance. Na⁺ conduction is readily distinguishable from K⁺ conduction in that it produces a steady-state negative conductance. The admittance of the Na⁺ system can show an anomalous resonance or an antiresonance depending on whether the net shunt conductance is negative or positive. Use of the Na⁺ negative conductance to neutralize leakage yields a measurement of dielectric capacitance at low frequency. A 90^o phase angle suggests that the capacitance is ideal.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Exponentiated exponential model (Gompertz kinetics) of Na+ and K+ conductance changes in squid giant axon

The conductance changes, gK(t) and gNa(t), of squid giant axon under voltage clamp (Hodgkin and Huxley, 1952) may be modeled by exponentiated exponential functions (Gompertz kinetics) from any holding potential VO to any membrane clamp potential V. The equation constants are set by the membrane potential V, and include, for any voltage step in the case of gK, the initial conductance, gO, the asymptote conductance g, and rate constant k: gK = g exp(-be-kt) where b = 1n g/gO. Equations of similar form relate g and k to the voltage V, and govern the corresponding parameters of the gNa system. For the gNa, the fast phase y = y exp (-be-kt) is cut down in proportion to a slow process p = (1 - p)e-k't + p, and thus gNa = py. The expo-exponential functions involve fewer constants than the Hodgkin-Huxley model. In particular, the role of the n, m, h parameters appears to be filled largely by 1n (g/gO) in the case of gK and by 1n (y/yO) in the case of gNa. Membrane action potentials during current clamp may be computed from the conductances generated by use of the appropriate differential forms of the equations; diverse other membrane behaviors may be predicted.     

Single ion occupancy and steady-state gating of Na channels in squid giant axon

The properties of the small fraction of tetrodotoxin (TTX)-sensitive Na channels that remain open in the steady state were studied in internally dialyzed voltage clamped squid giant axons. The observed Ussing flux ratio exponent (n') of 0.97 plus minus 0.03 (calculated from simultaneous measurements of TTX-sensitive current and (22)Na efflux) and nonindependent behavior of Na current at high internal [Na] are explained by a one-site ("1s") permeation model characterized by a single effective binding site within the channel pore in equilibrium with internal Na ions (apparent equilibrium dissociation constant K(Nai)(0) = 0.61 +/- 0.08 M). Steady-state open probability of the TTX-sensitive channels can be modeled by the product p(a)p(infinity), where p(a) represents voltage-dependent activation described by a Boltzmann distribution with midpoint V(a) = -7 mV and effective valence z(a) = 3.2 (Vandenberg, C.A., and F. Bezanilla. 1991. BIOPHYS: J. 60:1499--1510) coupled to voltage-independent inactivation by an equilibrium constant (Bezanilla, F., and C.M. Armstrong. 1977. J. Gen. Physiol. 70:549--566) K(eq) = 770. The factor p(infinity) represents voltage-dependent inactivation with empirical midpoint V(infinity)= -83 plus minus 5 mV and effective valence z(infinity) = 0.55 plus minus 0.03. The composite p(a)p(infinity)1s model describes the steady-state voltage dependence of the persistent TTX-sensitive current well.     

Single sodium channels from the squid giant axon

Since the work of A. L. Hodgkin and A. F. Huxley (1952. J. Physiol. [Lond.].117:500-544) the squid giant axon has been considered the classical preparation for the study of voltage-dependent sodium and potassium channels. In this preparation much data have been gathered on macroscopic and gating currents but no single sodium channel data have been available. This paper reports patch clamp recording of single sodium channel events from the cut-open squid axon. It is shown that the single channel conductance in the absence of external divalent ions is approximately 14 pS, similar to sodium channels recorded from other preparations, and that their kinetic properties are consistent with previous results on gating and macroscopic currents obtained from the perfused squid axon preparation.     

Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

Phosphorylation of K+ channels in the squid giant axon. A mechanistic analysis

Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In squid giant axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.     

Reversible electrical breakdown of squid giant axon membrane

Charge pulse relaxation experiments were performed on squid giant axon. In the low voltage range, the initial voltage across squid axon membrane was a linear function of the injected charge. For voltages of the order of 1 V this relationship between injected charge and voltage across the membrane changes abruptly. Because of a high conductance state caused by these large electric fields the voltage across the membrane cannot be made large enough to exceed a critical value, Vc, defined as the breakdown voltage, Vc has for squid axon membrane a value of 1.1 V at 12 degrees C. During breakdown the specific membrane conductance exceeds 1 S. cm-2. Electrical breakdown produced by charge pulses of few microseconds duration have no influence on the excitability of the squid axon membrane. The resealing process of the membrane is so fast that a depolarizing breakdown is followed by the falling phase of a normal action potential. Thus, membrane voltages close to Vc open the sodium channels in few microseconds, but do not produce a decrease of the time constant of potassium activation large enough to cause the opening of a significant percentage of channels in a time of about 10 mus. It is probable that the reversible electrical breakdown is mainly caused by mechanical instability produced by electrostriction of the membrane (electrochemical model), but the decrease in the Born energy for ion injection into the membrane, accompanying the decrease in membrane thickness, may play also an important role. Because of the high conductance of the membrane during breakdown it seems very likely that this results in pore formation.     

Nuclear magnetic resonance study of squid giant axon

Using a spin-echo technique, the spin-lattice and spin-spin relaxation times (T1 and T2) of water protons in a single nerve fiber (giant axon of squid) were determined. Similar measurements were also carried out on axoplasm extruded from these nerve fibers. It was found that the relaxation times of water protons of both the intact fiber and the extruded axoplasm are approximately equal (and much less than those of a free solution), suggesting that the relaxation times of cellular water are shortened mainly by water-protein interactions rather than by water-membrane interactions.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Transient polarization currents in the squid giant axon.

It has been repeatedly noted that the change of conformation of the molecules that serve as the ion-selective channels for sodium and potassium conductance in the nerve membrane will be accompanied by a change in the dipole moment of the molecule. This time-dependent change of dipole moment will produce transient currents in the membrane. The canonical form for these currents is determined with conventional statistical mechanics formalism. It is pointed out that the voltage dependence of the conductance channel conductance determines the free energy of the system to within a factor that is an unknown function of the voltage. Since the dipole currents do not depend on this unknown function, they are completely determined 0y the observed properties of the conductance system. The predicted properties of these dipole currents, their time constants and strengths, are calculated. By using the observed properties of gating currents, the density of the sodium channels is computed. The predicted properties of the dipole currents are found to compare satisfactorily with the observed properties of gating currents.     

Pressure dependence of the potassium currents of squid giant axon

Summary The effect of pressure upon the delayed, K, voltage-clamp currents of giant axons from the squidLoligo vulgaris was studied in axons treated with 300nm TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, V, of 31 cm³/mole at 15°C; V increased to about 42 cm³/mole at 5°C.Pressure slightly increased the magnitude, but did not produce any obvious major change in the voltage dependence, of the steady-state K conductance estimated from the current jump at the end of step depolarizations of small amplitude (to membrane potentials,E, 20 mV) and relatively short duration. At higher depolarizations, pressure produced a more substantial increase of the late membrane conductance, associated with an apparent enhancement of a slow component of the K conductance which could not be described within the framework of the Hodgkin-Huxley (HH)n ⁴ kinetic scheme.The apparent V values that characterize the pressure dependence of the early component of the K conductance are very close to those that describe the effect of pressure on Na activation kinetics, and it is conceivable that they are related to activation volumes involved in the isomerization of the normal K channels. The enhancement of the slow component of membrane conductance by pressure implies either a large increase in the conductance of the ionic channels that are responsible for it or a strong relative hastening of their turn-on kinetics.     

Anion conductances of the giant axon of squid Sepioteuthis.

Anion conductances of giant axons of squid, Sepioteuthis, were measured. The axons were internally perfused with a 100-mM tetraethylammonium-phosphate solution and immersed in a 100-mM Ca-salt solution (or Mg-salt solution) containing 0.3 microns tetrodotoxin. The external anion composition was changed. The membrane currents had a large amount of outward rectification due to anion influx across Cl- channels of the membrane (Inoue, 1985). The amount of outward rectification depended on the species of anion used and was strongly influenced by temperature and internal pH. In contrast to the anion conductances themselves, the conductance relative to Cl- (gA/gCl) was found to be quite stable against changes in the membrane potential, temperature, and pH. It is therefore suggested that each gA/gCl is an intrinsic quantity of the Cl- channel of the squid axon membrane. The sequence and values of gA/gCl obtained in this study were NO3- (1.80) greater than I- (1.40) greater than Br- (1.07) greater than Cl- (1.00) greater than MeSO3- (0.46) greater than H2PO2- (0.33) greater than CH3COO- (0.29) greater than SO4(2-) (0.06).     

Binding of radioactively labeled saxitoxin to the squid giant axon

Summary The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid,Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2–4°C of 4.3±1.7nm (meanse), and a linear, nonsaturable component. The density of saturable binding sites is 166±20.4 m^–2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977,J. Gen. Physiol. 70: 549). This single site conductance value of 7 pS is in close agreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons, and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.