K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane.

In the range of pH examined (5.2-10), variations of internal pH from high to low values result in a reversible decrease of the conductance of the open K channels, without significantly affecting the kinetics parameters. A linear plot of the conductance versus internal pH suggests the existence of a titratable group that has an apparent pKa of about 6.9, and that is accessible to protons only from the intracellular side of the membrane.     

Voltage-dependent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives

When giant axons of squid, Sepioteuthis, were bathed in a 100 mM Ca-salt solution containing tetrodotoxin (TTX) and internally perfused with a solution of 100 mM tetraethylammonium-salt (TEA-salt) or tetramethylammonium-salt (TMA-salt), the membrane potential was found to become sensitive to anions, especially Cl-. Membrane currents recorded from those axons showed practically no time-dependent properties, but they had a strong voltage-dependent characteristic, i.e., outward rectification. Cl- had a strong effect upon the voltage-dependent membrane currents. The nonlinear property of the currents was almost completely suppressed by some disulfonic stilbene derivatives applied intracellularly, such as 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are blockers of chloride transport. On the basis of these experimental results, it is concluded that a voltage-dependent chloride-permeable channel exists in the squid axon membrane. The chloride permeability (PCl) is a function of voltage, and its value at the resting membrane (Em = -60 mV) is calculated, using the Goldman-Hodgkin-Katz equation, to be 3.0 X 10(-7) cm/s.     

An analysis of conductance changes in squid axon

The membrane of the squid axon is considered on the basis of a pore model in which the distribution of the pore sizes strongly favors K⁺ transfer when there is no potential. Electrical asymmetry causes non-penetrating ions on the membrane capacitor to exert a mechanical force on both membrane surfaces and this force results in a deformation of the membrane pore system such that it assumes a distribution of sizes favoring the ions exerting mechanical force. The ions involved appear to be Ca⁺⁺ on the outside of the membrane and isethionate^-, (i^-) on the inside; as Ca⁺⁺ is equivalent in size to Na⁺, the charged membrane is potentially able to transfer Na⁺, when the ions deforming the membrane pore distribution are removed. A depolarization of the membrane leads to an opening of pores that will allow Na⁺ penetration and a release of the membrane from deformation. The pores revert to the zero-potential pore size distribution hence the Na permeability change is a transient. Calculation shows that the potassium conductance vs. displacement of membrane potential curve for the squid axon and the "inactivation" function, h, can be obtained directly from the assumed membrane distortion without the introduction of arbitrary parameters. The sodium conductance, because it is a transient, requires assumptions about the time constants with which ions unblock pores at the outside and the inside of the membrane.     

Frequency entrainment of squid axon membrane

Summary 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.     

Effects of barium on the potassium conductance of squid axon

Ba++ ion blocks K+ conductance at concentrations in the nanomolar range. This blockage is time and voltage dependent. From the time dependence it is possible to determine the forward and reverse rate constants for what appears to be an essentially first-order process of Ba++ interaction. The voltage dependence of the rate constants and the dissociation constants place the site of interaction near the middle of the membrane field. Comparison of the efficacy of Ba++ block at various internal K+ concentrations suggests that Ba++ is probably a simple competitive inhibitor of K+ interaction with the K+ conductance. The character of Ba++ block in high external K+ solutions suggests that Ba++ ion may be "knocked-off" the site by inward movement of external K+. Examination of the effects of other divalent cations suggests that the channel may have a closed state with a divalent cation inside the channel. The relative blockage at different temperatures implies a strong interaction between Ba++ and the K+ conductance.     

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.     

Noise measurements in squid axon membrane

Summary A small area (10^–4 to 10^–5 cm² patch) of the external surface of a squid (Loligo pealei) axon was isolated electrically by means of a pair of concentric glass pipettes and sucrose solution to achieve a low extraneous noise measurement of spontaneous fluctuations in membrane potential and current. The measured small-signal impedance function of the isolated patch in seawater was constant at low frequencies and declined monotonically at frequencies beyond 100 Hz. It is shown that the power-density spectrum (PDS) of voltage noise, which generally reflects the current-noise spectrum filtered by the membrane impedance function, is equivalent to the power spectrum of current-noise up to frequencies where the impedance decline is significant (Fishman, 1973a, Proc. Nat. Acad. Sci. USA 70:876). This result is in contrast to an impedance resonance measured under uniform constant-current (internal axial wire) conditions, for which the voltage-noise PDS reflects the impedance resonance. The overdamped resonance in the patch technique is a consequence of the relatively low resistance (1 M) pathways through the sucrose solution in the interstitial Schwann cell space which surround and shunt the high resistance (10–100 M) membrane patch. Current-noise measurements during patch voltage clamp extend observation of patch ionconductance fluctuations to 1 kHz. Various tests are presented to demonstrate the temporal and spatial adequacy of patch potential control during current-noise measurements.     

Light scattering spectroscopy of the squid axon membrane

Light scattering studies on the giant squid axon were done using the technique of optical mixing spectroscopy. This experimental approach is based on the use of laser light to detect the fluctuations of membrane macromolecules which are associated with conductance fluctuations. The light scattering spectra were similar to the Lorentzian-like behavior of conductance fluctuations, possibly reflecting an underlying conformational change in the specific membrane sites responsible for the potassium ion conductance. The amplitude of the spectra measured, increased when the membrane was depolarized and decreased on hyperpolarization. The spectra were fit to the sum of two terms, a (1/fcomponent and a simple Lorentzian term. Spectra from deteriorating axons did not show sensitivity to membrane potential changes. It is shown theoretically that fluctuations due to the voltage-dependent variable, n, of the Hodgkin-Huxley formalism are identical to the voltage fluctuations. The derived power spectrum is that of a second order system, capable of showing resonance peaking only if the voltage dependence of the potassium rate constants is included in the analysis. The lack of resonance peaking in the observed light scattering spectra, indicates that the data are best described by a damped second order system.     

Oscillatory behavior of the squid axon membrane potential

Squid axons impaled with a microelectrode have been treated with concentrations of xylene and benzene such that there is no change in threshold or resting potential at 20°C., while the spike height declines about 10 mv. A decrease in ambient temperature results in large, reversible, increases in threshold. While neither low temperature nor the added blocking agent induces repetitive firing from a single stimulus, the two treatments when combined do yield repetitive responses which commence at a sharply defined temperature. The alteration in the membrane responsible for the effects observed can be described by saying that there has been a large increase in the inductance of the equivalent electric circuit, and the temperature coefficient of the apparent membrane inductance has a Q₁₀ = 5.     

Extrinsic charge movement in the squid axon membrane

The absorption of the lipophilic anions dipycrilamine (DPA^-) and tetraphenylborate (TPhB^-) by the lipid matrix of the squid axon membrane, and the kinetics of their translocation, were studied by the charge pulse relaxation technique. The axons were treated with tetrodotoxin (TTX) and 4-aminopyridine to block the ionic currents responsible for nerve excitation. At high enough concentrations of absorbed ions ( 10^-12 mol/cm²) the membrane voltage relaxation following a brief current pulse consisted mainly of two exponential components, whose time constants and relative amplitudes were used for estimating the translocation rate constant, K, and the density of absorbed ions, N. These measurements were performed at different hydrostatic pressures in the range 1–100 MPa ( 1,000 atm), and at different temperatures in the range 5° C–20° C. Both K and N were found to be little affected by pressure. The pressure dependence of K indicated that the translocation of lipophilic ions across the nerve membrane involves activation volumes of the order of 5 cm³/mol. In all experiments the passive membrane resistance was little affected by pressures up to 80 MPa. However, above 100 MPa it fell dramatically to low values, presumably because of phase separation phenomena between the membrane components. The temperature dependence of K, both for DPa^- and TPhB^-, implied an activation energy for ion translocation of the order of 60 kJ/mol, close to that measured in artificial lipid bilayers.It is concluded that the lipid bilayer structure of the nerve membrane is not modified by pressures below 80 MPa and that the intramembrane movements of relatively small charged groups cannot account for the large activation volumes involved in the gating of ionic channels.     

Kinetics of activation of the potassium conductance in the squid giant axon

A quantitative re-investigation of the time course of the initial rise of the potassium current in voltage-clamped squid giant axons is described. The n4 law of the Hodgkin-Huxley equations was found to be well obeyed only for the smallest test pulses, and for larger ones a good fit of the inflected rise required use of the expression (1-exp[-t/tau n1])X-1(1-exp[-t/tau n2]), where both of the time constants and the power X varied with the size of the test pulse. Application of a negative prepulse produced a delay in the rise resulting mainly from an increase of X from a value of about 3 at -70 mV to 8 at -250 mV, while tau n1 remained constant and tau n2 was nearly doubled. The process responsible for generating this delay was switched on with a time constant of 8 ms at 4 degrees C, which fell to about 1 ms at 15 degrees C. Analysis of the inward tail currents at the end of a voltage-clamp pulse showed that there was a substantial external accumulation of potassium owing to the restriction of its diffusion out of the Schwann cell space, which, when duly allowed for, roughly doubled the calculated value of the potassium conductance. Computations suggested that the principal effect of such a build-up of [K]o would be to reduce the fitted values of tau n1 and tau n2 to two-thirds or even half their true sizes, while the power X would generally be little changed; but it would not affect the necessity to introduce a second time constant, nor would it invalidate our findings on the effect of negative prepulses.     

Noise measurements in squid axon membrane.

A small area (10(-4) to 10(-5) cm2 patch) of the external surface of a squid (Loligo pealei) axon was "isolated" electrically by means of a pair of concentric glass pipettes and sucrose solution to achieve a low extraneous noise measurement of spontaneous fluctuations in membrane potential and current. The measured "small-signal" impedance function of the isolated patch in seawater was constant at low frequencies and declined monotonically at frequencies beyond 100Hz. It is shown that the power-density spectrum (PDS) of voltage noise, which generally reflects the current-noise spectrum filtered by the membrane impedance function, is equivalent to the power spectrum of current-noise up to frequencies where the impedance decline is significant (Fishman, 1973a, Proc. Nat. Acad. Sci. USA 70:876). This result is in contrast to an impedance resonance measured under uniform constant-current (internal axial wire) conditions, for which the voltage-noise PDS reflects the impedance resonance. The overdamped resonance in the patch technique is a consequence of the relatively low resistance (1 Momega) pathways through the sucrose solution in the interstitial Schwann cell space which surround and shunt the high resistance (10-100 Momega) membrane patch. Current-noise measurements during patch voltage clamp extend observation of patch ion-conductance fluctuations to 1 kHz. Various tests are presented to demonstrate the temporal and spatial adequacey of patch potential control during current-noise measurements.     

Cation selectivity of the resting membrane of squid axon

Summary Permeability constant ratios among monovalent cations were studied in the resting membrane of a giant axon of a Pacific squid,Loligo opalescens, by observing the relationship between the membrane potential and the ion concentration.The average permeability ratios are: Tl, 1.8; K, 1.0; Rb, 0.72; Cs, 0.16; Na, <0.08; Li, <0.08. These permeability ratios suggest that neither valinomycin nor nonactin are adequate models for the sites producing the resting permeability in the axonal membrane.Cyclic polyetherbis(t-butyl cyclohexyl) 18-crown-6 does not increase the permeability ratioP _Cs/P _K except when applied at concentrations (5×10^–5 m) at which the surfactant properties of this molecule may become significant.     

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.     

Patch voltage clamp of squid axon membrane.

A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise.