Chemically induced K+ conduction noise in squid axon

Internal perfusion of tetraethylammonium ions (TEA) in squid axons produces a significant high frequency noise component. Although internal TEA suppresses the potassium conductance (G _K) noise at relatively low frequencies, it induces high frequency noise which exceeds the intensity of the normal potassium and sodium noise. In addition, the induced noise is dependent on the presence of internal potassium ions (K⁺) suggesting that this source of noise arises from a modulation of the K⁺ conductance due to the blocking and unblocking of the K⁺ channel. The simplest model describing the TEA data is a two-step sequential pseudo-unimolecular reaction where TEA binds during an open conductance state. A unit channel conductance of 2 pS is estimated from the TEA data as well as noise induced by triethyldecylammonium (TEDA) ions. Thus, these data are consistent with the hypothesis that the channel is blocked whenever the quaternary ammonium ion binding site, located near or within the K⁺ channel, is occupied.     

White noise measurement of squid axon membrane impedance.

The use of white noise techniques for system identification is illustrated by the following characterization of the subthreshold membrane impedance of the squid giant axon, space-clamped in a double sucrose gap. Power spectra were also computed. Depolarization increases the resonance, shifts the resonant frequently upward and decreases the membrane's inductive reactance. Reduced external Ca⁺⁺ increases the resonance, shifts the resonant frequency downward and increases the inductive reactance.     

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.     

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.     

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.     

Complex admittance of Na+ conduction in squid axon

Summary 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 dominateY(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^o phase angle suggests that the capacitance is ideal.     

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.     

Toward a multi-membrane model for potassium conduction in squid giant axon.

Possible physical mechanisms are considered which come close to a quantitative explanation for features of the potassium admittance magnitude. At 1–30 Hz there is an elevation of [Y] and positive phase above that obtained from the Hodgkin-Huxley model. Moreover there appears to be a slight negative phase for lower frequencies. An additional important feature for model fitting is the movement of the middle zero-phase crossing to the left with depolarization. Two general classes of subsystems are discussed. (1) Extracellular: potassium accumulation, barriers to diffusion near or adjacent to the excitable membrane, diffusion with volume flow, bulklimited diffusion through the Schwann cell layer and adsorption or absorption by the Schwann cells; (2) processes intrinsic to the excitable membrane: cyclic steady state, co-operative, inactivating and second order. A generalized potassium inactivation is treated in detail which provides fairly quantitative fits to transmembrane transfer data with a voltage-dependent inactivation time constant ranging between 40 and 100 ms. However, potassium accumulation coupled with hypothesized sorptive effects of the greater membrane, particularly the Schwann cell layer, also provide reasonable fits. Based on lack of experimental evidence for an inactivation, the choice is made for a multicompartment model. When an HH membrane element is combined with accumulation-depletion in an extracellular space and with a bulk limited or surface limited diffusion through the Schwann cells good agreement is obtained with measured admittance.     

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.     

Frequency domain analysis of asymmetry current in squid axon membrane.

The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.     

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.     

Injury-induced vesiculation and membrane redistribution in squid giant axon

Injury of isolated squid giant axons in sea water by cutting or stretching initiates the following unreported processes: (i) vesiculation in the subaxolemmal region extending along the axon several mm from the site of injury, followed by (ii) vesicular fusions that result in the formation of large vesicles (20-50 micron diameter), 'axosomes', and finally (iii) axosomal migration to and accumulation at the injury site. Some axosomes emerge from a cut end, attaining sizes up to 250 microns in diameter. Axosomes did not form after axonal injury unless divalent cations (Ca2+ or Mg2+) were present (10mM) in the external solution. The requirement for Ca2+ and the action of other ions are similar to that for cut-end cytoskeletal constriction in transected squid axons (Gallant, P.E. (1988) J. Neurosci. 8, 1479-1484) and for electrical sealing in transected axons of the cockroach (Yawo, H. and Kuno, M. (1985) J. Neurosci. 5, 1626-1632). Axosomes probably consist of membrane from different sources (e.g., axolemma, organelles and Schwann cells); however, localization of axosomal formation to the inner region of the axolemma and the formation dependence on divalent cations suggest principal involvement of cisternae of endoplasmic reticulum. Patch clamp of excised patches from axosomes liberated spontaneously from cut ends of transected axons showed a 12-pS K+ channel and gave indications of other channel types. Injury-induced vesiculation and membrane redistribution seem to be fundamental processes in the short-term (minutes to hours) that precede axonal degeneration or repair and regeneration. Axosomal formation provides a membrane preparation for the study of ion channels and other membrane processes from inaccessible organelles.     

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.     

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.     

Dynamics of potassium ion currents in squid axon membrane. A re-examination.

The original experiments of Cole and Moore (1960. Biophys. J. 1:161-202.), using conditioning and test membrane potentials to examine the dynamics of the potassium channel conductance in the squid axon, have been extended to test voltage levels by the use of tetrodotoxin to block the sodium conductance. The potassium currents for test voltage levels from -20 to +85 mV were superposable by translation along the time axis for all conditions tested: (a) with depolarizing conditioning voltages; (b) with hyperpolarizing conditioning voltages; and (c) in normal and in high potassium external media. The only deviations from superposition seen were when the internal sodium concentration was abnormally high and the potassium currents showed saturation at high levels of depolarization. Some restoration toward normal kinetics could be obtained by rapidly repeated depolarizations.     

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.