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.     

The effects of homologous series of anaesthetics on a resting potassium conductance of the squid giant axon

The effects of n-alkanes (n-pentane to n-octane), n-alkanols (n-pentanol to n-undecanol) and two carboxylic esters (methyl pentanoate and methyl octanoate) on the conductance of squid giant axons in a high potassium, zero sodium bathing solution have been examined. Sodium and delayed rectifier potassium channels were as far as possible pharmacologically blocked. A substantial fraction of the measured conductance is attributed to a recently-described, voltage-independent, potassium channel. Anaesthetics block this channel but its sensitivity is markedly different from those of other squid axon ion channels.     

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.     

Analysis of sodium current fluctuations in the cut-open squid giant axon

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.     

A series-parallel model of the voltage-gated sodium channel

A series-parallel model of the kinetics of the voltage-gated sodium channel is described. It goes some way towards reconciling the time-courses of the gating and macroscopic sodium currents in the squid giant axon with the molecular structure of the channel.     

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.     

Material from the Internal Surface of Squid Axon Exhibits Excess Noise: Implications in Modeling Membrane Noise

A fluid material from a squid (Loligo pealei) axon was isolated by mechanical application of two types of microcapillary (1-3-mum Diam) to the internal surface of intact and cut-axon preparations. Current noise in the isolated material exceeded thermal levels and power spectra were 1/f in form in the frequency range 1.25-500 Hz with voltage-dependent intensities that were unrelated to specific ion channels. Whether conduction in this material is a significant source of excess noise during axon conduction remains to be determined. Nevertheless, a source of excess noise external to or within an ion channel may not be properly represented solely as an additive term to the spectrum of ion channel noise; a deconvolution of these spectral components may be required for modeling purposes.     

Effects of the dipolar form of phloretin on potassium conductance in squid giant axons.

The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK(Em) and greater reductions maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; on postulates a phloretin "receptor" near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.     

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.     

Ion Channel Clustering at the Axon Initial Segment and Node of Ranvier Evolved Sequentially in Early Chordates

In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin–spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features (lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.     

CK2 accumulation at the axon initial segment depends on sodium channel Nav1

Accumulation of voltage-gated sodium channel Nav1 at the axon initial segment (AIS), results from a direct interaction with ankyrin G. This interaction is regulated in vitro by the protein kinase CK2, which is also highly enriched at the AIS. Here, using phosphospecific antibodies and inhibition/depletion approaches, we showed that Nav1 channels are phosphorylated in vivo in their ankyrin-binding motif. Moreover, we observed that CK2 accumulation at the AIS depends on expression of Nav1 channels, with which CK2 forms tight complexes. Thus, the CK2–Nav1 interaction is likely to initiate an important regulatory mechanism to finely control Nav1 phosphorylation and, consequently, neuronal excitability.     

Rapid sodium channel conductance changes during voltage clamp steps in squid giant axons.

The sodium conductance of the membrane of the giant axon of squid was isolated by the use of potassium-free solutions and voltage-clamped with pulses containing three levels of depolarization. The conductance appears to undergo rapid changes during certain repolarizing clamp steps whose voltage reach at least partially overlaps the gating range. The percentage change in conductance increases with time of depolarization from approximately 0 to approximately 25-30% at 7 ms for a potential step from +70 to -30 mV. Conductance steps were also observed for voltage steps from various depolarized levels to -70 mV. All observed shifts were in the direction of a decreased conductance. The conductance steps appear to be a weak function of the concentration of external calcium, which also acts as a voltage-dependent channel blocker for inwardly directed sodium currents. A number of possible mechanisms are suggested. One of these is discussed in some detail and postulates a voltage- and time-dependent molecular process that does not itself yield open or closed channel conformations, but that affects the magnitude of the rate constants that do connect open and closed state conformations.     

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

Peripheral nerve at extreme low temperatures 2: Pharmacologic modulation of temperature effects

Changes in temperature have profound and clinically important effects on the peripheral nerve. In a previous paper, the effects of temperature on many properties of the peripheral nerve action potential (NAP) were explored including the NAP amplitude, conduction velocity and response to paired pulse stimulation. In this paper, the effects of pharmacologic manipulations on these parameters were explored in order to further understand the mechanisms of these effects.The reduction in conduction velocity with temperature was shown to be independent of the ionic composition of the perfusate and was unaffected by potassium or sodium channel blockade. This implies that the phenomenon of reduced conduction velocities at low temperature may be related to changes in the passive properties of the axon with temperature. Blockade of sodium channels and chronic membrane depolarization produced by high perfusate potassium concentrations or high dose 4-aminopyridine impair the resistance of the nerve to hypothermia and enhance the injury to the nerve produced by cycles of cooling and rewarming. This suggests the possibility that changes in the sodium inactivation channel may be responsible for the changes in the NAP amplitude with temperature and that prolonged sodium inactivation may lead more permanent changes in excitability.     

Collective Equilibrium Behaviour of Ion Channel Gating in Cell Membranes: An Ising Model Formulation

A statistical mechanical model for voltage-gated ion channels in cell membranes is proposed using the transfer matrix method. Equilibrium behavior of the system is studied. Representing the distribution of channels over the cellular membrane on a one-dimensional array with each channel having two states (open and closed) and incorporating channel–channel cooperative interactions, we calculate the fraction of channels in the open state at equilibrium. Experimental data obtained from batrachotoxin-modified sodium channels in the squid giant axon, using the cut-open axon technique, is best fit by the model when there is no interaction between the channels.     

Identified ion channels in the squid nervous system

Our modern understanding of channels as discrete voltage-sensitive and ion-selective entities comes largely from a series of classical studies using the squid giant axon. This system has also been critical for understanding how transporters and synaptic transmission operate. This review outlines attempts to assign molecular identities to the extensively studied physiological properties of this system. As it turns out, this is no simple task. Molecular candidates for voltage-gated Na(+), K(+), and Ca(2+) channels, as well as ion transporters have been isolated from the squid nervous system. Both physiological and molecular approaches have been used to equate these cloned gene products with their native counterparts. In the case of the delayed rectifier K(+) conductance, the most thoroughly studied example, two major issues further complicate the equation. First, the ability of K(+) channel monomers to form heteromultimers with unique properties must be considered. Second, squid K(+) channel mRNAs are extensively edited, a process that can generate a wide variety of channel proteins from a common gene. The giant axon system is beginning to play an important role in understanding the biological relevance of this latter process.     

The Cole-Moore Effect: Still Unexplained?

In the first issue, on the first page of the Biophysical Journal in 1960, Cole and Moore provided the first confirmation of the Hodgkin and Huxley formulation of the sodium and potassium conductances that underlie the action potential. In addition, working with the squid giant axon, Cole and Moore noted that strong hyperpolarization preceding a depolarizing voltage-clamp pulse delayed the rise of the potassium conductance: once started, the time course of the rise was always the same but after significant hyperpolarization there was a long lag before the rise began. This phenomenon has come to be known as the Cole-Moore effect. Their article examines and disproves the hypothesis that the lag reflects the time required to refill the membrane with potassium ions after the ions are swept out of the membrane into the axoplasm by hyperpolarization. The work by Cole and Moore indirectly supports the idea of a membrane channel for potassium conductance. However, the mechanism of the Cole-Moore effect remains a mystery even now, buried in the structure of the potassium channel, which was completely unknown at the time.     

An approach to the study of intracellular proteins related to the excitability of the squid giant axon

The technique for covalently labeling proteins with 125I-labelled Bolton-Hunter reagent was used to determine the quantities of proteins released from the axoplasmic side of the squid axon membrane. The reagent could be introduced into the interior of the axon by the technique of intracellular perfusion, the radioiodination reaction being carried out in situ. Alternatively, the reaction could be carried out in vitro, i.e., by mixing the reagent with samples of proteins dissolved in the intracellular perfusion fluid collected from the axon. This technique was found to be sensitive enough to permit analysis of a large number of protein samples collected from a single axon. By the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis, it was found that proteins of approx. 56 000 daltons were released into the perfusate when a solution of potassium chloride or potassium bromide was introduced into the interior of an axon. Suppression of axonal excitability was associated with this release of proteins. The significance of these findings in relation to the structure and function of the axon is discussed.     

On the voltage-dependent action of tetrodotoxin.

The use of the maximum rate-of-rise of the action potential (Vmax) as a measure of the sodium conductance in excitable membranes is invalid. In the case of membrane action potentials, Vmax depends on the total ionic current across the membrane; drugs or conditions that alter the potassium or leak conductances will also affect Vmax. Likewise, long-term depolarization of the membrane lessens the fraction of total ionic current that passes through the sodium channels by increasing potassium conductance and inactivating the sodium conductance, and thereby reduces the effect of Vmax of drugs that specifically block sodium channels. The resultant artifact, an apparent voltage-dependent potency of such drugs, is theoretically simulated for the effects of tetrodotoxin on the Hodgkin-Huxley squid axon.