Sodium currents in the giant axon of the crab Carcinus maenas

Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crab Carcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10-150 microsec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 microsec at -40 mV (at 18 degrees C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at -70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than -100 mV lock a fraction of the Na channels in a closed conformation.     

Potassium currents in the giant axon of the crab Carcinus maenas

Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crab Carcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol, (London) 143:76-90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using the m3h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standard n4 Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

Monovalent cation permeabilities of the potassium systems in the crab giant axon

Summary Permeability ratios for pairs of monovalent cations permeating the two potassium systems proposed for the giant axon of the crabCarcinus maenas (M. E. Quinta-Ferreira, E. Rojas & N. Arispe,J. Membrane Biol. 66:171–181, 1982b) were estimated from measurements of the reversal potential of the currents under voltage-clamp conditions. With K⁺ inside the axon, permeability ratios from the reversal potential of the currents through the late channel are:P _Rb/P _K=0.9, /P _K<0.2 andP _Cs/P _K=0.18. With Cs⁺ inside the ratios are:P _K/P _Cs=8.7,P _Rb/P _Cs=7.1 and /P _Cs=2.4. The analysis of the inward currents carried by Rb⁺ or NH ₄ ⁺ showed similar reversal potentials for the early transient component and the late sustained component. Whence, the sequence of permeabilities for the two types of potassium channels is:P _K>P _Rb> >P _Na=P _Cs. The time constants for the activation of the two components recorded either in K-, Rb-, or NH₄-artificial seawater are twice as large as the corresponding time constants measured in Na-artificial seawater.     

Differential blockage of two types of potassium channels in the crab giant axon

Summary Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crabsCarcinus maenas andCancer pagirus. The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate types of potassium channels exist in these axons (M. E. Quinta-Ferreira, E. Rojas and N. Arispe,J. Membrane Biol. 66:171–181, 1982). It is shown here that, with small concentrations of conventional K⁺-channel blockers, it is possible to differentially inhibit these channels. The potassium channels with activation and fast inactivation gating (m³h, Hodgkin-Huxley kinetics) were blocked by external application of 4 amino-pyridine (4-AP). The potassium channels with standard gating (n⁴, Hodgkin-Huxley kinetics) were preferentially inhibited by externally applied tetraethylammonium (TEA). The differential blockage of the two types of potassium conductance changes suggests that they represent two different populations of potassium channels.It is further shown here that blocking the early transient conductance increase leads to the inhibition of the repetitive electrical activity induced by constant depolarizing current injection in fibers fromCardisoma guanhumi.     

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.     

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.     

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.     

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.     

Potassium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetic and steadystate characteristics of the potassium conductance in the giant axon of the crabCarcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol. (London) 143:76–90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using them ³ h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standardn ⁴ Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

Sodium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crabCarcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10–150 sec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 sec at –40 mV (at 18°C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at –70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than –100 mV lock a fraction of the Na channels in a closed conformation.     

Synaptic potential in the motor giant axon of the crayfish

Some electrical properties of the synapses between central giant axons (presynaptic) and the motor giant axon (postsynaptic) of the crayfish abdominal nerve cord have been investigated. Postsynaptic potential change in response to presynaptic volleys contains two components: a spike potential and a synaptic potential of very long time course. Amplitude of the synaptic potential is graded according to the number of active presynaptic axons. Conductance increase in the synaptic membrane endures over most of the period of potential change, and it is this rather than the "electrical time constant" of the membrane that in large measure determines the form of the synaptic potential. Temporal summation of synaptic potential occurs during repetitive presynaptic stimulation, and after such stimulation the rate of decay of synaptic potential is greatly slowed.     

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.     

Electrical coupling and dye transfer between axon segments in the medium giant axon of the earthworm

Electrical coupling between axon segments has been studied in the medium giant axon, which had been partially isolated from the ventral nerve cord of the earthworm. Evidence has been obtained that in addition to the coupling structures in the septum there are diffusion- and current-pathways through the pseudo-myelin which seem to be more permeable to fluorescein than the channels in the septum. These findings offer an explanation for the discrepancy of the experimentally determined specific resistance of the septum and the expected values based on nexus density and channel size in the septum.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Insect-like olfactory adaptations in the terrestrial giant robber crab

The robber crab (Birgus latro), also known as the coconut crab, is the world's largest land-living arthropod, with a weight reaching 4 kg and a length of over half a meter [1]. Apart from the marine larval stage 2, 3, this crab is fully terrestrial, and will actually drown if submerged in water [4]. A transition from sea to land raises dramatically new demands on the sensory equipment of an animal. In olfaction, the stimulus changes from hydrophilic molecules in aqueous solution to mainly hydrophobic in the gaseous phase [5]. The olfactory system of land crabs thus represents an excellent opportunity for investigating the effects of the transition from sea to land. Have land crabs come to the same solutions as other terrestrial animals, or is their olfactory sense characterized by unique innovations? Here, we show that the robber crab has evolved an olfactory sense with a high degree of resemblance to the insect system. The similarities extend to physiological, behavioral, and morphological characters. The insect nose of the robber crab is a striking example of convergent evolution and nicely illustrates how similar selection pressures result in similar adaptation     

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.     

Electrical phenomena in nerve; squid giant axon

The action of a number of agents, which may be classified as "stabilizers" and "unstabilizers" on the electrical oscillations and after-potentials in the squid giant axon has been examined. The effects on the spike, "positive overshoot," and "potassium potential" were also observed, but where possible concentrations were employed which left these phenomena unaltered. Veratrine augmented the oscillations and the negative after-potential, particularly with repetitive stimulation. Yohimbine caused a small long lasting positive after-potential and depressed the oscillations, effects also enhanced with repetitive activity. Cocaine and procaine suppressed the oscillations and the negative after-potential but DDT was completely inert. An elevation in the medium calcium depressed the oscillations and the naturally occurring negative after-potential; negative after-potentials induced with veratrine were increased by calcium. A decrease in the potassium augmented the oscillations and the negative after-potential. A hypothesis is presented in which these effects are interpreted in terms of potassium concentration at the fiber surface as regulated by a labile permeability and metabolism. This is discussed in relation to the available evidence for these factors. It is a pleasure to acknowledge the author's indebtedness to Dr. D. E. S. Brown, Director, and to his staff at the Bermuda Biological Station for Research for the cooperation and special facilities provided during the initiation of this work. Dr. T. Baylor of Princeton University very kindly provided the camera and film used in Bermuda.     

Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [³H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)⁺ RNA in all tissue samples. Radioactive poly(A)⁺ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)⁺ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.Special Issue dedicated to Prof. Holger Hydén.