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Complex admittance of Na+ conduction in squid axon.

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 dominate Y(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 degrees phase angle suggests that the capacitance is ideal.

Animals↗

Cytoplasmic gel and water relations of axon.

A previous method of measuring the swelling pressure (delta IIg) of the cytoplasmic gel of the giant axon of Loligo vulgaris was refined. The estimates of delta IIg made with the improved method were consistent with those made with the earlier method. In these methods the activity of the solvent in the gel is measured by increasing the activity of the solvent in the internal phase of the gel by application of hydrostatic pressure to the gel directly. Comparable values for the activity of the solvent in the gel were obtained also by an alternate method, in which the deswelling of the gel is measured upon decreasing the activity of the solvent in the external phase by addition of a nonpenetrating high mol wt polymer (i.e., Ficoll). Additional support was obtained for the earlier suggestion that delta IIg contributes to the swelling and shrinkage pattern of the whole axon. In part, the new evidence involved two consecutive direct measurements of intraxonal pressure. The first measurement was that of a mixed pressure composed of delta IIg and delta IIm (delta IIm being the effective osmotic pressure due to the intra-extraxonal gradient in the activity of mobile solutes). The subsequent measurement was that of delta IIg alone. The latter measurement was made feasible by destroying the axolemma, thereby eliminating the contribution of delta IIm. An estimate of delta IIm was obtained by subtracting delta IIg from the total pressure measured initially. The delta IIm determined by the above method was two orders of magnitude smaller than the theoretical osmotic pressure. This is consistent with the delta IIm determined previously, where osmotic intra-extraxonal filtration coefficients were compared to the hydrostatic. The mixed pressure experiments lend credence to the idea that the substantial contribution of delta IIg to the water relations of the whole axon is due to delta IIg being of the same order of magnitude as delta IIm. The degree of free swelling of axoplasmic gels was studied as a function of pH, salt concentration, and hydration radius of the anion of the salt used. The swelling increased with an increase in the reciprocal of the hydration radius, a decrease in salt concentration, and at pH below or above similar to 4.5. The nature of the constraints to the free swelling of axoplasm in axons immersed in seawater was studied. With the seawater employed, these constraints appeared to be due more to the retractive forces of the sheath than to delta IIm.

Animals↗

Slowing of the time course of the excitation of squid giant axons in viscous solutions.

The time course of excitation of intracellularly perfused squid giant axons was slowed as the solution viscosity was raised by adding neutral molecules, i.e., glucose and glycerol. By twofold increase of the solution viscosity, the duration of action potential was prolonged to 2.7-fold and the maximum rate of rise decreased to one-half. At the same time, the membrane resistance at resting state increased by 60%. These effects were reversible. The time course of inward and outward currents was slowed also. When the solution viscosity increased to twofold, the time to peak inward current increased by 80%, and the amplitudes of peak inward and steady outward currents decreased by 60% and by 70%, respectively. These effects were not specific for the sodium or the potassium channel. Effects of solution viscosity occurred in both hypotonic and hypertonic solutions. Q10 values of temperature dependence of the time course of the action potential were equal in any viscous solutions. These effects in viscous solutions were explained by the change in solution viscosity but not by the change in solution osmolarities, ionic activities, or solution resistivity.

Action Potentials↗

Membrane capacity of squid giant axon during hyper- and depolarizations.

The change in membrane capacitance and conductance of squid giant axons during hyper- and depolarizations was investigated. The measurements of capacitance and conductance were performed using an admittance bridge with resting, hyperpolarizaed and depolarized membranes. The duration of DC pulses is 20-40 msec and is long enough to permit the admittance measurements between 1 and 50 kHZ. The amplitudes of DC pulses were varied between 0 and 40 mV for both depolarization and hyperpolarization. Within these limited experimental conditions, we found a substantial increase in membrane capacitance with depolarization and a decrease with hyperpolarization. Our results indicate that the change in membrane capacitance will increase further if low frequencies are used with larger depolarizing pulses. The change in membrane capacitance is frequency dependent and it increases with decreasing frequencies. The analyses based on an equivalent circuit (vide infra) gives rise to a time constant of active membrane capacitance close to that of sodium currents. This result indicates that the observed capacitance changes may arise from sodium channels. A brief discussion is given on the nature of frequency-dependent membrane capacitance of nerve axons.

Animals↗

Effects of fatty acids on membrane currents in the squid giant axon.

The effects of fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular and extracellular application of the test substances. Fatty acids mainly suppress the Na current but have little effect on the K current. These effects are completely reversed after washing with control solution. The concentrations required to suppress the peak inward current by 50% and Hill number were determined for each fatty acid. ED50 decreased about 1/3 for each increase of one carbon atom. The standard free energy was -3.05 kJ mole-1 for CH2. The Hill number was 1.58 for 2-decenoic acid. The suppression effect of the fatty acids depends on the number of carbon atoms in the compounds and their chemical structure. Suppression of the Na current was clearly observed when the number of carbon atoms exceeded eight. When fatty acids of the same chain length were compared, 2-decenoic acid had strong inhibitory activity, but sebacic acid had no effect at all on the Na channel. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (J. Physiol. (London) 117:500-544, 1952) and the changes in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m infinity) for the Na current against membrane potential and the time constant of activation (tau m) were shifted 20 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation (tau h) was almost no change by application of the fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Local anesthetic action of carboxylic esters: evidence for the significance of molecular volume and for the number of sites involved.

The effects of the homologous series of carboxylic esters, methyl propionate to methyl decanoate, on the steady-state inactivation of the sodium current in squid axons have been studied. The esters moved the relationship between the inactivation parameter, h infinity, and the membrane potential in the hyperpolarizing direction, thus reducing the number of sodium channels available at the resting potential. The concentration dependence of the shift at the mid-point of the curve of h infinity against potential has been measured for all esters except decanoate, which was almost inactive. Two aspects of these concentration dependences suggest that molecular volume is an important determinant of the effectiveness of each ester. Firstly, there is a sharp decline in activity above methyl hexanoate. This cut-off in activity resembles that for hydrocarbons where it has been suggested [e.g., Haydon, D.A., Urban, B.W. 1983, J. Physiol. (London) 341:411-427] to a result from a decrease in uptake with increasing molecular volume. (Further data for the hydrocarbons n-butane to n-heptane are reported here.) Secondly, the smallest compounds, methyl propionate and methyl butyrate, are less effective than would be predicted if equal membrane concentrations of each ester produced the same shift. The aqueous concentration dependences for these esters indicate that below methyl hexanoate, as the series is descended, progressively higher membrane concentrations are required to produce a given shift. This would be expected if the volume of ester in the membrane, rather than the number of molecules, is important.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthetics, Local↗

D2O and the sodium pump in squid nerve membrane.

In 10 K artificial seawater (ASW), D2O replacement reduced the Na efflux of squid axons by about one third. In 0 K ASW, D2O replacement had little effect. D2O reduced the K+ sensitivity of the efflux but increased the affinity for K+. A 4 degrees decrease in temperature mimicked the effects of D2O. When axons were injected with arginine, to decrease the ATP/ADP ratio, they lost K+ sensitivity in normal ASW, as expected. Their efflux into 0 K ASW became D2O sensitive. The results are discussed in terms of conformational changes in the Na pump molecular complex.

Animals↗

Frequency entrainment of squid axon membrane.

Sinusoidally varying stimulating currents were applied to space-clamped squid giant axon membranes in a double sucrose gap apparatus. Stimulus parameters varied were peak-to-peak current amplitude, frequency, and DC offset bias. In response to these stimuli, the membranes produced action potentials in varying patterns, according to variation of input stimulus parameters. For some stimulus parameters the output patterns were stable and obviously periodic with the periods being simple multiples of the input period; for other stimulus parameters no obvious periodicity was manifest in the output. The experimental results were compared with simulations using a computer model which was modified in several ways from the Hodgkin-Huxley model to make it more representative of our preparation. The model takes into account K+ accumulation in the periaxonal space, features of Na+ inactivation which are anomalous to the Hodgkin-Huxley model, sucrose gap hyperpolarization current, and membrane current noise. Many aspects of the experiments are successfully simulated but some are not, possibly because some very slow process present in the preparation is not included in the model.

Action Potentials↗

Cation permeability ratios of sodium channels in normal and grayanotoxin-treated squid axon membranes.

Permeabilities of squid axon membranes to various cations at rest and during activity have been measured by voltage clamp before and during internal perfusion of 4 X 10(-5) M grayanotoxin I. The resting sodium and potassium permeabilities were estimated to be 6.85 X 10(-8) cm/sec and 2.84 X 10(-6) cm/sec, respectively. Grayanotoxin I increased the resting sodium permeability to 7.38 10(-7) cm/sec representing an 11-fold increase. The potassium permeability was increased only by a factor of 1.24. The resting permeability ratios as estimated by the voltage clamp method before application of grayanotoxin I were Na (1): Li (0.83): formamidine (1.34): guanidine (1.49): Cs (0.87): methylguanidine (0.86): methylamine (0.78). Grayanotoxin I did not drastically the resting permeability ratios with a result of Na (1): Li (0.95): formamidine (1.27): guanidine (1.16): Cs (0.47): methylguanidine (0.72): methylamine (0.46). The membrane potential method gave essentially the same resting permability ratios before and during application of grayanotoxin I if corrections were made for permeability to choline as the cation substitute and for changes in potassium permeability caused by test cations. The permeability ration choline/Na was estimated to be 0.72 by the voltage clamp method and 0.65 by the membrane potential method. Grayanotoxin I decreased the ration to 0.43. The permeability ratios during peak transient current were estimated to be Na (1): Li (1.12): formamidine (0.20): guanidine (0.20): Cs (0.085): methylguanidine (0.061): methylamine (0.036). Thus the sodium channels for the peak current are much more selective to cation than the resting sodium channels. It appears that the resting sodium channels in normal and grayanotoixn I-treated axons are operationally different from the sodium channels that undergo a conductance increase upon stimulation.

Animals↗

Binding of radioactively labeled saxitoxin to the squid giant axon.

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 degrees C of 4.3 +/- 1.7 nM (mean SE), and a linear, nonsaturable component. The density of saturable binding sites is 166 +/- 20.4 micrometers-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 aggreement 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.

Animals↗

Changes in absorption, fluorescence, dichroism, and Birefringence in stained giant axons: : optical measurement of membrane potential.

The absorption, fluorescence, dichroism, and birefringence of stained squid axons were measured during action potentials and voltage clamp steps in an effort to find large optical signals that could be used to monitor membrane potential. Changes in all four optical properties were found that were linearly related to membrane potential and, with several new dyes, the signal-to-noise ratios were larger than any obtained previously. The problem of photodynamic damage was greatly diminished; with a merocyaninerhodanine dye, the photodynamic damage associated with intense light and the presence of oxygen was negligible. The absorption change obtained with this dye was relatively large; it could be measured with a signal-to-noise ratio of 100:1 during a single action potential.

Action Potentials↗

ATP-Dependent chloride influx into internally dialyzed squid giant axons.

Measurements were made of 36C1 influx into squid giant axons whose internal solutes were controlled by means of internal dialysis. When the intracellular chloride concentration was 50mM and the internal concentration of adenosine 5'-triphosphate (ATP) was 4mM, the average chloride influx was 11.6 pmoles/cm2 x sec. When the axons were dialyzed with an ATP-free solution, the average influx fell to 5.1 pmoles/cm2 x sec. The effect was fully reversible upon the return of ATP to the dialysis fluid. Chloride-36 influx in the presence and absence of ATP was found to be inversely related to the internal chloride concentration.

Adenosine Triphosphate↗

Measurements of amino acid transport in internally dialyzed giant axons.

It is shown that the axoplasmic composition of acidic and neutral amino acids can be controlled effectively by the method of internal dialysis. Direct assay for specific binding and measurement of diffusion coefficients in axoplasm show that there is no significant binding or compartmentalization of amino acids. The dependence of amino acid efflux on substrate concentration can be measured under well-defined, true steady-state conditions. The taurine efflux-concentration relation in the Myxicola giant axon conforms to a second-order Hill equation. This fact is consistent with either a cooperative process or a mechanism in which membrane translocation is not the rate-controlling step. The effluxes of taurine and glycine from squid axon are an order of magnitude smaller than in Myxicola. The efflux-concentration relations are essentially linear up to 200 mM substrate concentration. This result may be produced by specific transporters which have very high asymmetry, or by simple diffusive leak in the absence of specific transporters.

Amino Acids↗