Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.     

Ionic conductances of squid giant fiber lobe neurons

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.     

Cultured Giant Fiber Lobe of Squid Expresses Three Distinct Potassium Channel Activities in Selective Combinations

Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane. Received: 24 October 1995/Revised: 5 March 1996     

Depolarization-induced slowing of Ca2+ channel deactivation in squid neurons.

Properties of squid giant fiber lobe (GFL) Ca2+ channel deactivation (closing) were studied using whole-cell voltage clamp. Tail currents displayed biexponential decay, and fast and slow components of these tails exhibited similar external Ca(2+)- and voltage-dependence. Both components also shared similar inactivation properties. Increasing duration pulses to strongly depolarizing potentials caused a substantial slowing of the rate of deactivation for the fast component, and also led to an apparent conversion of fast tail currents to slow without an increase in total tail amplitude. A five-state kinetic model that computed the closing of channels differentially populating two open states could simulate the kinetic characteristics of GFL Ca2+ pulse and tail currents over a wide voltage range. The kinetics of the proposed state transition was very similar to the time course of relief of omega-Agatoxin IVA Ca2+ channel block with long pulses. A similar model predicted that the relief of block could occur via faster toxin dissociation from the second open state. Thus, GFL Ca2+ channels possess a unique form of voltage-dependent gating modification, in which maintained prior depolarization leads to a significant delay to channel closure at negative potentials. At the nerve terminal, amplified Ca2+ signals generated by such a mechanism might alter synaptic responses to repetitive stimulation.     

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.     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

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.     

Characterization of the ATP-promoted aspect of Na+-Ca2+ exchange present in squid retinal nerve axolemma

Using an in vitro system which consists of an axolemma-rich vesicle fraction prepared from squid retinal nerve fibers, an Na+-Ca2+ exchange process has been characterized and appears identical with that reported in squid giant axon. This exchange is absolutely dependent on the establishment of an Na+ gradient, shows monovalent and divalent cation specificity and is highly sensitive to monensin, A23187 and valinomycin but not to ouabain, digitoxigenin, vanadate, pentylenetetrazole, tetrodotoxin or tetraethylammonium. Furthermore, it was found that the exchange process is enhanced by the addition of ATP. This ATP-promoted aspects of Na+-Ca2+ exchange shares many similar characteristics with Na+-Ca2+ ATP hydrolysis and may indicate a common mechanism for both activities via a protein phosphorylation-dephosphorylation event.     

Pharmacological and kinetic analysis of K channel gating currents

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.     

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.     

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.     

Differentiated pattern of sodium channel expression in dissociated Purkinje neurons maintained in long-term culture

Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

The compensation of potential changes produced by trivalent erbium ion in squid giant axon with applied potentials.

The transmembrane potential of voltage-clamped squid giant axon is increased to compensate for a reduction in the rate of potassium channel kinetics when artificial seawater with trivalent erbium ion is substituted for artificial seawater. The additional potential required to produce an equivalent rise time is a measure of the potential shift produced by the erbium ions. When the kinetics of K+ channels are matched in this manner, the maximal K+ currents are larger for the larger transmembrane potential. This observation requires a functional separation of the open K+ channel and the voltage sensor for the gating mechanism of this channel.     

Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization

We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

A comparative study of the effects of tetrodotoxin and the removal of external Na+ on the resting potential: Evidence of separate pathways for the resting and excitable Na currents in squid axon

To investigate whether the Na permeability of the resting membrane is determined predominantly by the excitable Na channel, we examined the effects of tetrodotoxin (TTX) and the complete removal of external Na+ on the resting potential. In the intact squid axon bathed in K-free artificial seawater, both TTX and the removal of Na+ produced small hyperpolarizations. The effect of Na removal, however, was larger than that of TTX. In the perfused squid axon, the hyperpolarization produced by the removal of external Na+ was greatly enhanced when the internal K concentration ([K+]i) was reduced. The effect of TTX, on the other hand, was not sensitive to the [K+]i or to the membrane potential. For [K+]i = 50 mM and [K+]o = 0, the average hyperpolarization produced by TTX was 1.2 mV, while the hyperpolarization produced by Na removal was approximately 21 mV. The difference between these two effects suggests that the majority of the resting Na current passes through pathways other than the excitable Na channel.     

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.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.