Ionic Current Measurements in the Squid Giant Axon Membrane

The concepts, experiments, and interpretations of ionic current measurements after a step change of the squid axon membrane potential require the potential to be constant for the duration and the membrane area measured. An experimental approach to this ideal has been developed. Electrometer, operational, and control amplifiers produce the step potential between internal micropipette and external potential electrodes within 40 microseconds and a few millivolts. With an internal current electrode effective resistance of 2 ohm cm.², the membrane potential and current may be constant within a few millivolts and 10 per cent out to near the electrode ends. The maximum membrane current patterns of the best axons are several times larger but of the type described by Cole and analyzed by Hodgkin and Huxley when the change of potential is adequately controlled. The occasional obvious distortions are attributed to the marginal adequacy of potential control to be expected from the characteristics of the current electrodes and the axon. Improvements are expected only to increase stability and accuracy. No reason has been found either to question the qualitative characteristics of the early measurements or to so discredit the analyses made of them.     

Membrane Current Noise in Lobster Axon under Voltage Clamp

Random fluctuations in the steady-state current of neural membrane were measured in the giant lobster axon by means of a low noise voltage-clamp system. The power density spectrum S(f) of the fluctuations was evaluated between 20 and 5120 Hz and found to be of the type 1/f. Mean values of the potassium, sodium, and leakage currents Ī_K, Ī_Na, and Ī_L were also measured by usual voltage-clamp techniques. Comparisons between these two types of data recorded under a number of different experimental conditions, such as presence of tetrodotoxin (TTX), substitution of calcium by lanthanum, and changes in the external concentration of potassium, have strongly suggested that the intensity of the fluctuations is related to the magnitude of Ī_K.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

Sodium and Potassium Ion Effluxes from Squid Axons under Voltage Clamp Conditions

Squid giant axons loaded with Na²⁴ were subjected to short duration (0.5 msec.) clamped depolarizations of about 100 mv at frequencies of 20/sec. and 60/sec. while in choline sea water. Under such conditions the early outward current was just about maximal at the time of termination of the clamping pulse. An integration of the early current versus time record gave 1.2 μcoulomb/cm² pulse, while a measurement of the extra Na²⁴ efflux resulting from repetitive pulsing gave a charge transfer of 1.4 μcoulomb/cm² pulse. In sodium-containing sea water and with pulses 50-75 mv more positive than E_Na the Na²⁴ efflux is about 3 times the measured charge transfer. The efflux of K⁴² from a previously loaded axon into normal sea water is only 50 per cent of the measured charge transfer when the membrane is held for about 5 msec. at a potential such that there is no early current, and such pulses are at 10-20/sec. The experiments appear to confirm the suggestion that the early current during bioelectric activity is sodium but provide unsatisfactory support for the identification of the delayed but sustained current solely with potassium ions. Resting Na⁺ efflux is 0.6 pmole/cm² sec. mmole [Na]₁, while the apparent K⁺ efflux is about 250 pmole/cm² sec. and is little affected by hyperpolarization.     

Ammonium Ion Currents in the Squid Giant Axon

Voltage-clamp studies on intact and internally perfused squid giant axons demonstrate that ammonium can substitute partially for either sodium or potassium. Ammonium carries the early transient current with 0.3 times the permeability of sodium and it carries the delayed current with 0.3 times the potassium permeability. The conductance changes observed in voltage clamp show approximately the same time course in ammonium solutions as in the normal physiological solutions. These ammonium ion permeabilities account for the known effects of ammonium on nerve excitability. Experiments with the drugs tetrodotoxin (TTX) and tetraethyl ammonium chloride (TEA) demonstrate that these molecules block the early and late components of the current selectively, even when both components are carried by the same ion, ammonium.     

An Analysis of the Membrane Potential along a Clamped Squid Axon

A partially depolarized squid axon membrane is assumed to have a quasi-steady state negative resistance, the membrane potential is clamped at one point, and a distribution of potential along the axon is obtained from the cable equation. Nominal experimental values of -2 ohm cm² for the membrane and 6 ohm cm² for the internal and external current electrodes and the axoplasm and sea water between them are used for illustration. The potential and current may be uniform for an axon and electrode length less than 1.2 mm. For a long axon the potential varies as the cosine of the distance within 0.8 mm of the control point. Beyond this the potential variation is exponential and the entire pattern is about 5 mm long. The average current density out to 0.3 mm from the control point is within 10 per cent of the potential clamp value. These distributions are stable for control amplifications of about unity and more.     

Permeability of Squid Axon Membrane to Various Ions

The permeability of the squid axon membrane was determined by the use of radioisotopes of Na, K, Ca, Cs, and Br. Effluxes of these isotopes were measured mainly by the method of intracellular injection. Measurements of influxes were carried out under continuous intracellular perfusion with an isotonic solution of potassium sulfate. The Na permeability of the resting (excitable) axonal membrane was found to be roughly equal to the K permeability. The permeability to anion was far smaller than that to cations. It is emphasized that the axonal membrane has properties of a cation exchanger. The physicochemical nature of the "two stable states" of the excitable membrane is discussed on the basis of ion exchange isotherms.     

Kinesin mRNA Is Present in the Squid Giant Axon

Abstract: Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding β-actin and β-tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.     

An Analysis of the Surface Fixed-Charge Theory of the Squid Giant Axon Membrane

The observed shift in threshold potential, after perfusion of the squid giant axon with solutions of low ionic strength, can be predicted by assuming a fixed negative charge on the inside of the membrane. The constant field equation, together with the double-layer potential due to this charge, has been used to determine the change in resting potential during perfusion with solutions of low ionic strength. Neither the modified constant field equation nor Planck's diffusion equation can successfully predict the observed shift in resting potential. It is suggested that a positive charge distribution exists about the sodium channel on the outside of the membrane. The double-layer potential due to this positive charge, together with the independence principle, has been used to predict the relationship between sodium current and membrane potential when the ionic strength and sodium activity of the external solution are decreased. These predictions have been compared with the available experimental observations.     

Slow Changes of Potassium Permeability in the Squid Giant Axon

A slow potassium inactivation i.e. decrease of conductance when the inside of the membrane is made more positive with respect to the outside, has been observed for the squid axon. The conductance-potential curve is sigmoid shaped, and the ratio between maximum and minimum potassium conductance is at least 3. The time constant for the change of potassium conductance with potential is independent of the concentration of potassium in the external solution, but dependent upon potential and temperature. At 9 degrees C and at the normal sea water resting potential, the time constant is 11 sec. For lower temperature or more depolarizing potentials, the time constant is greater. The inactivation can be described by modifying the Hodgkin-Huxley equation for potassium current, using one additional parameter. The modified equation is similar in form to the Hodgkin-Huxley equation for sodium current, suggesting that the mechanism for the passive transport of potassium through the axon membrane is similar to that for sodium.     

Organization of the Cortical Endoplasmic Reticulum in the Squid Giant Axon

The organization of the cortical endoplasmic reticulum in the squid giant axon was investigated by rapid freeze and freeze-substitution electron microscopy, thereby eliminating the effects of fixatives on this potentially labile structure. Juvenile squid, which have thinner Schwann sheaths, were used in order to achieve freezing deep enough to include the entire axonal cortex. The smooth endoplasmic reticulum is composed of subaxolemmal and deeper cisternae, tubules, tethers and vesicles. The subaxolemmal cisternae make junctional contacts with the axolemma which are characterized by filamentous-granular bridging structures approximately 3 nm in diameter. The subaxolemmal junctions with the axolemma resemble the coupling junctions between the sarcoplasmic reticulum and the T-tubules in muscle. Reconstruction of short series of sections showed that a number of the elements of the endoplasmic reticulum were continuous but numerous separate vesicles were present as well. The morphology of endoplasmic reticulum as described here suggests that it is a highly dynamic entity as well as a Ca²⁺ sequestering organelle.     

Uptake of Protein by the Giant Axon of the Squid

THE uptake of macromolecules by neurones and glia may be relevant to an understanding of the relationships between these cells and to the concept of the glia-neurone unit¹. Protein uptake has been studied biochemically using suspensions of animal cells², but little morphological evidence has been gathered from neuronal systems^3–5. This problem can be investigated conveniently using the giant axon of the squid; biochemical analyses can be carried out on samples of axoplasm after incubation of intact axons with a labelled macromolecule. To do this we have used¹²⁵I-albumin as a model protein because of its commercial availability and the lack of reincorporation of free ¹²⁵I-tyrosine into protein².     

Potassium Ion Current in the Squid Giant Axon: Dynamic Characteristic

Measurements of the potassium current in the squid axon membrane have been made, after changes of the membrane potential to the sodium potential of Hodgkin and Huxley (HH), from near the resting potential, from depolarizations of various durations and amplitudes, and from hyperpolarizations of up to 150 mv. The potassium currents I given by I = I_∞ {1 - exp [- (t + t₀)/τ]}²⁵, where t₀ is determined by the initial conditions, represent the new data and approximate the HH functions in the regions for which they are adequate. A corresponding modification for the sodium current does not appear necessary. The results support the HH assumptions of the independence of the potassium and sodium currents, the dependence of the potassium current upon a single parameter determined by the membrane potential, and the expression of this parameter by a first order differential equation, and, although the results drastically modify the analytical expressions, they very considerably extend the range of apparent validity of these assumptions. The delay in the potassium current after severe hyperpolarization is used to estimate a potassium ion mobility in the membrane as 10^-5 of its value in aqueous solutions.     

Subthreshold Behavior and Phenomenological Impedance of the Squid Giant Axon

The oscillatory behavior of the cephalopod giant axons in response to an applied current has been established by previous investigators. In the study reported here the relationship between the familiar "RC" electrotonic response and the oscillatory behavior is examined experimentally and shown to be dependent on the membrane potential. Computations based on the three-current system which was inferred from electrical measurements by Hodgkin and Huxley yield subthreshold responses in good agreement with experimental data. The point which is developed explicitly is that since the three currents, in general, have nonzero resting values and two currents, the "Na" system and the "K" system, are controlled by voltage-dependent time-variant conductances, the subthreshold behavior of the squid axon in the small-signal range can be looked upon as arising from phenomenological inductance or capacitance. The total phenomenological impedance as a function of membrane potential is derived by linearizing the empirically fitted equations which describe the time-variant conductances. At the resting potential the impedance consists of three structures in parallel, namely, two series RL elements and one series RC element. The true membrane capacitance acts in parallel with the phenomenological elements, to give a total impedance which is, in effect, a parallel R, L, C system with a "natural frequency" of oscillation. At relatively hyperpolarized levels the impedance "degenerates" to an RC system.     

Characterization of the Resting Axolemma in the Giant Axon of the Squid

Previous electron microscope studies have shown that the Schwann cell layer is traversed by long and tortuous slit-like channels ~60Å wide, which provide the major route of access to the axolemma surface. In the present work the restriction offered by the resting axolemma to the passage of six small non-electrolyte molecules has been determined. The radii of the probing molecules were estimated from constructed molecular models. The ability of the axolemma to discriminate between the solvent (water) and each probing molecule was expressed in terms of the reflection coefficient σ. σ was then used to calculate an effective pore size for the resting axolemma. The value of 4.25 Å found for the pore radius is in excellent agreement with the 1.5 to 8.5 Å limiting values previously calculated from our measurements of water fluxes. The presence of pores with 4.25 Å radius in the resting axolemma is compatible with restricted diffusion of Na. The present paper leads to the conclusion that the axolemma is the only continuous barrier across which the ionic gradient responsible for the normal functioning of the nerve can be maintained. The combined findings of electron microscopy, water permeability, and molecular restricted filtration indicate that in all probability the axolemma is the "excitable membrane" of the physiologists.