Derivatives of saxitoxin

The preparation, spectral characterization, chromatographic and biological properties of several derivatives of saxitoxin, a highly potent neurotoxin, are described. Derivatives include decarbamoylsaxitoxin, both epimers of dihydrosaxitoxin (α- and β-saxitoxinol) and both epimers of decarbamoylsaxitoxinol, as well as several isotopically labeled variants. Natural toxin is readily regenerated from these C13 and C12 alcohols by carbamoylation or oxidation procedures. Evaluation of the biological properties of these compounds provides information on the structural features required for toxin binding to the sodium channel. Hemisuccinate esters of decarbamoylsaxitoxin and saxitoxinol have also been prepared, and the latter derivative was coupled to bovine serum albumin to provide a conjugate for possible antibody production.     

Development of saxitoxin-conjugated affinity gels

Saxitoxin (STX) and its analogues accumulated in bivalves cause food poisoning through the blockade of sodium channels in the nervous system. In the current studies, STX-conjugated agarose gels as affinity chromatography reagents were prepared for investigation of the fate of the toxins in natural environments and in the human body. A carboxyl moiety was introduced through positions C11 and C13 to leave the most characteristic part of the molecule intact. Two types of synthesized derivatives, 11-(2-carboxyethylthio)saxitoxin and 13-O-hemisuccinyldecarbamoylsaxitoxin, were successfully conjugated to Sepharose 4B in high yield. Affinity gels containing 500 nmol of STX or decarbamoylsaxitoxin per milliliter of gel were accomplished by masking the residual amino groups by acetylation. Finally, the STX-conjugated affinity gel was effective for concentrating STX-binding proteins from pufferfish and bullfrog plasma.     

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon’s neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia–axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

Effects of bath resistance on action potentials in the squid giant axon: myocardial implications.

This study presents a simplified version of the quasi-one-dimensional theory (Wu, J., E. A. Johnson, and J. M. Kootsey. 1996. A quasi-one-dimensional theory for anisotropic propagation of excitation in cardiac muscle. Biophys. J. 71:2427-2439) with two components of the extracellular current, along and perpendicular to the axis, and a simulation and its experimental confirmation for the giant axon of the squid. By extending the one-dimensional core conductor cable equations, this theory predicts, as confirmed by the experiment, that the shapes of the intracellular and the extracellular action potentials are related to the resistance of the bath. Such a result was previously only expected by the field theories. The correlation between the shapes of the intracellular and the extracellular potentials of the giant axon of the squid resembles that observed during the anisotropic propagation of excitation in cardiac muscle. Therefore, this study not only develops a quasi-one-dimensional theory for a squid axon, but also provides one possible factor contributing to the anisotropic propagation of action potentials in cardiac muscle.     

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.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

Hydrogen sulfide as a precursor for the synthesis of isethionate in the squid giant axon.

Cysteine is taken up by the squid giant axon to about 200% of equivalent distribution, whereas sulfide is taken up (probably as hydrogen sulfide) to about 40% of equivalence. Thereafter, the squid axon synthesizes its major anion, isethionate, in about equal amounts from the sulfide, or from the sulfur of cysteine, but not at all from the carbons of cysteine. Squid nerve also contains rhodanese, an enzyme which transfers the outer (sulfane) sulfur of thiosulfate to cyanide to produce thiocyanate. It is speculated that, instead of “detoxifying cyanide,” as the reaction involving rhodanese is commonly described, the physiological role of this enzyme is the formation of a carbon-sulfur bond, leading finally, in the squid, to the formation of isethionate. This is the first evidence concerning the pathway for the synthesis of isethionate in squid nerve where this compound is normally present at a concentration of 150 mm.     

Periaxonal K+ regulation in the small squid Alloteuthis. Studies on isolated and in situ axons.

A novel giant axon preparation from the squid Alloteuthis is described. Properties of in situ and isolated axons are similar. Periaxonal K+ accumulation is a function of the physiological state of the animal and of the axon and its sheathing layers. Carefully dissected isolated axons, and axons in situ in a healthy mantle, show much less K+ accumulation than previously reported in squid. It is suggested that the Schwann cells are involved in the observed K+ regulation.     

Axolinin Localization in the Nervous Tissue of Squid Revealed by Monoclonal Antibodies Specific for Axolinin: Cellular and Subcellular Localization of Axolinin in the Squid Neuron

Cellular and subcellular distributions of axolinin, the 260-kilodalton (kD) microtubule-associated glycoprotein originally purified from squid axons, in various squid tissues such as optical lobes, bundles of small nerve fibers (fin nerves), giant stellate ganglia, skin, muscle, liver, and gill, were immunologically studied using monoclonal antibodies specifically recognizing the polypeptide chain of axolinin. The following results were obtained: (1) Axolinin is confined to squid neurons and skin; (2) axolinin is localized in the axon whereas another 260-kD microtubule-associated protein, MAP B, is localized in the cell bodies; and (3) axolinin is localized mainly in the peripheral part of the axoplasm of the squid giant axon. The last result has confirmed our previous conclusion obtained using polyclonal antisera against axolinin, which contain antibodies recognizing not only axolinin-specific epitopes but also nonspecific epitopes. The physiological importance of the localization of axolinin in axons and the skin is discussed based on its possible relationship to excitability function.     

Demonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride

1. Intracellular injection of tetraethylammonium chloride (TEA) into a giant axon of the squid prolongs the duration of the action potential without changing the resting potential (Fig. 3). The prolongation is sometimes 100-fold or more. 2. The action potential of a giant axon treated with TEA has an initial peak followed by a plateau (Fig. 3). The membrane resistance during the plateau is practically normal (Fig. 4). Near the end of the action potential, there is an apparent increase in the membrane resistance (Fig. 5D and Fig. 6, right). 3. The phenomenon of abolition of action potentials was demonstrated in the squid giant axon treated with TEA (Fig. 7). Following an action potential abolished in its early phase, there is no refractoriness (Fig. 8). 4. By the method of voltage clamp, the voltage-current relation was investigated on normal squid axons as well as on axons treated with TEA (Figs. 9 and 10). 5. The presence of stable states of the membrane was demonstrated by clamping the membrane potential with two voltage steps (Fig. 11). Experimental evidence was presented showing that, in an "unstable" state, the membrane conductance is not uniquely determined by the membrane potential. 6. The effect of low sodium water was investigated in the axon treated with TEA (Fig. 12). 7. The similarity between the action potential of a squid axon under TEA and that of the vertebrate cardiac muscle was stressed. The experimental results were interpreted as supporting the view that there are two stable states in the membrane. Initiation and abolition of an action potential were explained as transitions between the two states.     

An autoregulation model in the system of squid axonal channels

A model that describes the interactions in the system of squid axon channels is proposed. The variables of the model are channel concentration and membrane voltage. The model represents the simplest mathematical formulation of the data on axon membrane, its components, and their role in the generation and propagation of action potential.     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

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.     

No Conventional Function for the Conventional Kinesin?

A paper by DeGiorgis et al. (DeGiorgis JA, Petukhova TA, Evans TA, Reese TS. Kinesin-3 is an organelle motor in the squid giant axon. Traffic 2008; DOI: 10.1111/j.1600-0854.2008.00809.x) in this issue of Traffic reports on the identification and function of a second squid kinesin, a kinesin-3 motor. As expected, the newly discovered motor associates with axoplasmic organelles in situ and powers motility along microtubules of vesicles isolated from squid axoplasm. Less expected was the finding that kinesin-3 may be the predominant motor for anterograde organelle movement in the squid axon, which challenges the so far undisputed view that this function is fulfilled by the conventional kinesin, kinesin-1. These novel findings let us wonder what the real function of kinesin-1--the most abundant motor in squid axons--actually is.     

A Computer Evaluation of Equations for Predicting the Potential across Biological Membranes

The Goldman, Henderson, and Planck junction potential equations can be used to describe the potential across the resting giant squid axon. These equations are used to calculate the relative Na, K, and Cl permeabilities of the squid axon using the experimental measurements of Hodgkin and Katz. The equations all provide excellent agreement with the observed data and yield similar permeability values.     

A source of large axons for neurophysiology: the North Atlantic squid Illex illecebrosus.

A new axon preparation from the ommastrephid squid (Illex illecebrosusus (Lesueur)) is described. This squid is common in the Northwestern Atlantic and features a number of long, unbranched, and moderately large-diameter axons having no apparent 'twigging.' Although the diameters of these axons are somewhat smaller than those of 'giant' axons coming from some other species of squid, this axon preparation should be considered as an attractive alternative for neurobiologic research.     

Binding of radioactively labeled saxitoxin to the squid giant axon

Summary 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°C of 4.3±1.7nm (meanse), and a linear, nonsaturable component. The density of saturable binding sites is 166±20.4 m^–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 agreement 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.     

Control of the spatial distribution of sodium channels in giant fiber lobe neurons of the squid

Na+ channels are present at high density in squid giant axon but are absent from its somata in the giant fiber lobe (GFL) of the stellate ganglion. GFL cells dispersed in vitro maintain growing axons and develop a Na+ channel distribution similar to that in vivo. Tunicamycin, a glycosylation inhibitor, selectively disrupts the spatially appropriate, high level expression of Na+ channels in axonal membrane but has no effect on expression in cell bodies, which show low level, inappropriate expression in vitro. This effect does not appear to involve alteration in Na+ channel turnover or axon viability. K+ channel distribution is unaffected. Thus, glycosylation appears to be involved in controlling Na+ channel localization in squid neurons.