Molecular events that underlie membrane excitation.
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Action Potentials↗
Biomedical subjects
I Tasaki
Publications and source records attributed to I Tasaki.
Tetanic contraction of the crab nerve evoked by repetitive stimulation.
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Action Potentials↗
Further studies of rapid mechanical changes in squid giant axon associated with action potential production.
Mechanical changes in the squid giant axon associated with the production of an action potential are examined further by using piezoelectric and optical methods. The peak of swelling of the axon coincides with the peak of the action potential recorded internally at the site of mechanical recording. Mechanical changes produced by a train of action potentials do not summate. Repetitively fired action potentials induced by lowering the external Ca-ion concentration are preceded by a gradual swelling of the axon. An inward current through the membrane causes shrinkage and an outward current produced swelling of the axon. An inward current enhances and an outward current depresses the mechanical changes associated with the action potential. There is a transient shortening followed by an elongation of the axon when an action potential travels along the axon. It is argued that the experimental results obtained are consistent with the colloid chemical, or macromolecular, theory of excitation.
Action Potentials↗
Rapid pressure changes and surface displacements in the squid giant axon associated with production of action potentials.
By using both optical and mechano-electric detectors, we have shown that the squid giant axon swells when an action potential is generated. The maximum swelling is reached at the peak of the action potential. The undershoot of the membrane potential is associated with a marked shrinkage of the axon. We have also demonstrated these mechanical changes in axons from which a major portion of the axoplasm has been removed. We have examined the effects of changing the tonicity of the external medium and of applying several chemical reagents.
Action Potentials↗
Temperature changes associated with nerve excitation: detection by using polyvinylidene fluoride film.
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Animals↗
Removal of the Schwann sheath from the giant nerve fiber of the squid: an electron-microscopic study of the axolemma and associated axoplasmic structures.
The axolemma is associated structurally and functionally with the axoplasm, forming an axolemma-ectoplasm complex. To study the structure of this complex, a new technique was developed for removing the Schwann sheath from a portion of the giant nerve fiber. An isolated fiber was treated, without loss of excitability, with trypsin dissolved in natural seawater. Next, the fiber was treated with a mild fixative and then was placed in a hypertonic solution of sucrose in seawater. The elevated sheath was transected and everted, thus exposing the surface of the axon. Desheathed axons were examined by scanning and transmission electron microscopy. The surface of the axon has a ridge-and-groove pattern, reflecting an underlying helical arrangement of filaments which bundle and unbundle. Both left and right axons of the squid possess right-handed helical twists with a tilt angle of 10 degrees. Hemispherical protuberances about 1.5 microns at their base are observed along the ridges. Thin sections of the desheathed axons reveal that the desheathing procedure leaves the axolemma intact. Desheathed axons display electron-dense bodies associated with the axolemma and with the filaments of the ectoplasm similar to the dense bodies observed in whole fibers fixed in the presence of 10 mM Co(II) ions. Axons perfused for 40 min with a solution containing 2 mM Co(II) ions retain their excitability and display a smooth inner ectoplasmic face. A portion of the axolemma, together with adhering ectoplasm, was removed from desheathed axons, mounted between folding double grids, stained, and critical-point dried. Through this novel method a network of 10 nm filaments spaced 40 nm apart and cross-linked by filaments 5 to 7 nm in diameter was demonstrated.
Animals↗
Rapid mechanical changes in crab nerve and squid axon during action potentials.
1. By using a Fotonic sensor, it was found possible to record rapid mechanical responses of the crab nerve without a signal averager. 2. In squid giant axons, the peak of swelling was shown to coincide fairly accurately with the peak of the action potential. 3. Associated with propagation of an action potential along a squid giant axon, there is a small decrease followed by an increase in the length of the axon. 4. It was emphasized that both mechanical and birefringence responses of the squid axon are diphasic. 5. The origin of the mechanical responses in axons is discussed on the basis of the theories proposed by LOEB, HOBER and TEORELL.
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Swelling of nerve fibers associated with action potentials.
Swelling of nerve fibers during the action potential was demonstrated by three different methods. Generation of a propagated nerve impulse in a crab nerve produced an outward movement of 50 to 100 angstroms of the nerve surfce and a rise in swelling pressure on the order of 5 dynes per square centimeter. In squid giant axons, the amplitude of the observed outward movement of the surface was small.
Action Potentials↗
Mechanical changes in squid giant axons associated with production of action potentials.
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Action Potentials↗
Shortening of nerve fibers associated with propagated nerve impulse.
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Action Potentials↗
Synthesis and study of conformationally defined enantiomers of local anesthetics and conformationally defined enantiomers of fluorescent dyes designed to label electrically excitable membranes.
Conformationally defined enantiomeric local anesthetics and fluorescent dyes were synthesized. Neither the local anesthetics nor the fluorescent probes showed stereospecificity in interacting with nerve membranes. The fluorescence signals generated by the dyes showed excellent correlation with the time course and shape of the nerve action potential.
Action Potentials↗
Mechanical changes in crab nerve fibers during action potentials.
A nerve impulse travelling along a crustacean nerve was found to be accompanied by a small, rapid movement of the nerve surface. The movement was 10-20 nm in amplitude and was concurrent with a rise in the "swelling pressure" of the order of 5 mg/cm2 for a nerve bundle. Initiation of an action potential at the site of cathodal polarization was preceded by a small, slow mechanical change in the nerve fiber. Anodal polarization produced a large mechanical change of the opposite sign. Tetrodotoxin and procaine suppressed rapid mechanical changes.
Action Potentials↗
Swelling of nerve fibers during action potentials.
Nerve fibers swell concomitantly with the initiation of an action potential. Pronounced shrinkage follows the phase of swelling. These mechanical changes are attributed to the movement of water predicted by Teorell.
Action Potentials↗
Divalent cation dependent phosphorylation of proteins in squid giant axon.
In vitro and in situ (after intracellular infusion) incubation of axoplasm from the squid giant axon with [gamma-32P]ATP produces a phosphorylation of primarily two proteins (of mol.wt. 200,000 and greater than 400,000). The phosphorylation of these proteins is stimulated by Mg2+, inhibited by Ca2+, and unaffected by 10(-7) to 10(-5) M cyclic nucleotides. The 200 kdalton and greater than 400 kdalton phosphorylated peaks appear to be neurofilament proteins, and phosphorylation of these peaks in situ is decreased by electrical stimulation of the axon.
Adenosine Triphosphate↗
Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon.
Proteins in the squid giant axon were labeled with 32P by in vitro incubation of isolated axoplasm with radioactive [gamma-32P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400,000 and 200,000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [gamma-32P]ATP was applied in situ intracellularly or extracellarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.
Adenosine Triphosphate↗
Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization.
The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive 125-I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-aminopyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, aparticular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.
Action Potentials↗
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.
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Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability.
A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.
Action Potentials↗