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.     

Pressure dependence of sodium gating currents in the squid giant axon

Asymmetric displacement currents, I_g, were measured in squid axons at different hydrostatic pressures, P, up to 60 MPa. Potassium and sodium currents were abolished by intracellular Cs⁺ and TEA⁺, by extracellular Tetrodotoxin (TTX), and by Na⁺ substitution with Tris⁺. The time course of Ig became progressively slower with increasing pressure, and the amplitude decreased. With appropriate scaling in time and amplitude, I_g records at any given P could be made to superimpose very well with those obtained at atmospheric pressure. The same scaling factors yielded a good superposition of all records obtained for voltage steps to membrane potentials in the range-30 to +42 mV. The ratio between the amplitude and time factors was larger than unity and increased with P, indicating a progressive decrease (up to 35% at 60 MPa) of the total charge displaced, Q, with no significant change in its voltage dependence. The time-scaling factor increased exponentially with P, as expected if all the steps involved in the opening of a sodium channel, and producing a major charge redistribution, have the same activation volume, V _g 17 cm³/mol. This value is roughly one-half of that characterizing the pressure dependence of sodium current activation, suggesting that some late, rate-limiting step in the opening of sodium channels has a large activation volume without being accompanied by an easily detected charge movement.Part of the decrease of Q with pressure could be attributed to an increase in sodium inactivation. However, we cannot exclude the possibility that there is a reversible reduction in the number of fast activating sodium channels, similar to the phenomenon that has been reported to occur at low temperatures (Matteson and Armstrong 1982).     

Analysis of sodium current fluctuations in the cut-open squid giant axon

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.     

Kinetic analysis of the sodium gating current in the squid giant axon

A critical study has been made of the characteristics of the kinetic components of the sodium gating current in the squid giant axon, of which not less than five can be resolved. In addition to the principal fast component Ig2, there are two components of appreciable size that relax at an intermediate rate, Ig3 alpha and Ig 3 beta. Ig3 alpha has a fast rise, and is present over the whole range of negative test potentials. Ig3 beta is absent below -40 mV, exhibits a delayed onset and disappears on inactivation of the sodium system. There are also two smaller components, Ig1 and Ig4, with very fast and much slower relaxation time constants, respectively.     

The early phase of sodium channel gating current in the squid giant axon

A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligo forbesii. This component is characterized by relaxation time constants typically shorter than 25 µs. The charge displaced accounts for about 10% (or 2 nC/cm²) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above + 20 mV and below – 100 mV This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of 1 e^– and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.     

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.     

The effect of tetrodotoxin on the sodium gating current in the squid giant axon.

The effect of tetrodotoxin (TTX) on the sodium gating current in the squid giant axon was examined by recording the current that flowed at the pulse potential at which the ionic current fell to zero, first in the absence and then in the presence of TTX. The addition of 1 microM TTX to the bathing solution had no consistent effect on the size of the initial peak of the gating current, but resulted in small changes in the timecourse of its subsequent relaxation which were mainly caused by a reduction of about one quarter in the component that has a delayed onset and may possibly arise from changes in the state of ionization of groups in the channel wall when the lumen fills with water. Our findings suggest that the binding of TTX at the outer face of the sodium channel does not interfere with the mechanisms of activation and inactivation by the voltage sensors, but has an allosteric effect on the access of internal cations to the inside of the channel.     

Neurofilament protein is phosphorylated in the squid giant axon

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.     

Anesthetic and calcium action in the voltage-clamped squid giant axon

Changes in spike configuration and in the inward and outward currents of voltage-clamped axons agree in indicating that the increases in permeability to sodium and potassium ions during activity are depressed by procaine and cocaine and augmented by calcium. At low levels of depolarization, the effect of the multivalent ion is similar to that of the local anesthetics, in keeping with their similar effects on the threshold of excitability. The reduction of membrane conductance at rest requires a higher concentration of the drugs than that needed to affect the increase in permeability with activity.     

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.     

Kinetics of ion movement in the squid giant axon

The loss of Na²², K⁴², and Cl³⁶ from single giant axons of the squid, Loligo pealii, following exposure to an artificial sea water containing these radioisotopes, occurs in two stages, an initial rapid one followed by an exponential decline. The time constants of the latter stage for the 3 ion species are, respectively, 290, 200, and 175 minutes. The outflux of sodium is depressed while that of potassium is accelerated in the absence of oxygen; the emergence of potassium is slowed by cocaine, while that of sodium is unaffected. One cm. ends of the axons take up about twice as much radiosodium as the central segment; this difference in activity is largely preserved during exposure to inactive solution. Such marked differences are not observed with radiopotassium. From the experimental data estimates are given of the influxes and outfluxes of the individual ions. The kinetics of outflux suggests a cortical layer of measureable thickness which contains the ions in different proportions from those in the medium and which governs the rate of emergence of these ions from the axon as though it contained very few but large (relative to ion dimensions) pores.     

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.     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

On the slowly rising phase of the sodium gating current in the squid giant axon.

High-resolution records of the sodium gating current in the squid giant axon demonstrate the existence of a slowly rising phase that is first apparent at pulse potentials slightly below zero, and becomes increasingly pronounced at more positive potentials. At +80 mV the current reaches its peak with a delay of 30 microseconds at 10 degrees C. It is suggested that this current is generated by the first two steps labelled R-->P and P-->A in the S4 units of all four domains of the series-parallel gating system, activating the channel before its opening by the third steps A-->B in domains I, II and III in conjunction with hydration. The kinetics of the slowly rising phase can only be explained by the incorporation of an appropriate degree of voltage-dependent cooperativity between the S4 voltage-sensors for their two initial transitions.