Inactivation of the sodium permeability in squid giant nerve fibres.
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Animals↗
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Sodium inactivation and drug-induced immobilization of the gating charge in nerve membrane.
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Aconitine↗
Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon.
Cytoplasmic filaments, separated from the axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, support the directed movement of organelles in the presence of ATP. All organelles, regardless of size, move continuously along isolated transport filaments at 2.2 +/- 0.2 micron/sec. In the intact axoplasm, however, movements of the larger organelles are slow and saltatory. These movements may reflect a resistance to movement imposed by the intact axoplasm. The uniform rate of all organelles along isolated transport filaments suggests that a single type of molecular motor powers fast axonal transport. Organelles can attach to and move along more than one filament at a time, suggesting that organelles have multiple binding sites for this motor.
Adenosine Triphosphate↗
Single microtubules from squid axoplasm support bidirectional movement of organelles.
Single filaments, dissociated from the extruded axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, transport organelles bidirectionally. Organelles moving in the same or opposite directions along the same filament can pass each other without colliding, indicating that each transport filament has several tracks for organelle movement. In order to characterize transport filaments, organelle movements were first examined by video microscopy, and then the same filaments were examined by electron microscopy after rapid-freezing, freeze-drying, and rotary-shadowing. Transport filaments that supported bidirectional movement of organelles are 22 nm to 27 nm in diameter and have a substructure indicative of a single microtubule. Immunofluorescence showed that virtually all transport filaments contain tubulin. These results show that single microtubules can serve as a substratum for organelle movement, and suggest that an interaction between organelles and microtubules is the basis of fast axonal transport.
Animals↗
Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon.
A reconstituted system for examining directed organelle movements along purified microtubules has been developed. Axoplasm from the squid giant axon was separated into soluble supernatant and organelle-enriched fractions. Movement of axoplasmic organelles along MAP-free microtubules occurred consistently only after addition of axoplasmic supernatant and ATP. The velocity of such organelle movement (1.6 micron/sec) was the same as in dissociated axoplasm. The axoplasmic supernatant also supported movement of microtubules along a glass surface and movement of carboxylated latex beads along microtubules at 0.5 micron/sec. The direction of microtubule movement on glass was opposite to that of organelle and bead movement on microtubules. The factors supporting movements of microtubules, beads, and organelles were sensitive to heat, trypsin, AMP-PNP and 100 microM vanadate. All of these movements may be driven by a single, soluble ATPase that binds reversibly to organelles, beads, or glass and generates a translocating force on a microtubule.
Adenosine Triphosphate↗
Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro.
Single microtubules from squid axoplasm support bidirectional movement of organelles. We previously purified a microtubule translocator (kinesin) that moves latex beads in only one direction along microtubules. In this study, a polar array of microtubules assembled off of centrosomes in vitro was used to demonstrate that kinesin moves latex beads from the minus to the plus ends of microtubules, a direction that corresponds to anterograde transport in the axon. A crude solubilized fraction from squid axoplasm (S1a), however, generates bidirectional movement of beads along microtubules. Retrograde bead movement (1.4 micron/sec) is inhibited by N-ethylmaleimide and 20 microM vanadate while anterograde movement (0.6 micron/sec) is unaffected by these agents. Furthermore, a monoclonal antibody against kinesin, when coupled to Sepharose, removes the anterograde, but not the retrograde, bead translocator from S1a. These results indicate that there is a retrograde bead translocator which is pharmacologically and immunologically distinct from kinesin.
Animals↗
Enzymatic degradation of chitins and partially deacetylated chitins.
The enzymatic (lysozyme, chitinase etc.) digestibility of chitins obtained from squid pen and shrimp shell, and of partially deacetylated chitins (DA-chitins) was investigated. The digestibility of various chitins by the chitinase from Bacillus sp. PI-7S was much higher than that by lysozyme, and beta-chitin was digested more smoothly than alpha-chitin. DA-chitin deacetylated under homogeneous conditions (DAC) was hydrolysed by lysozyme more rapidly than that deacetylated under heterogeneous conditions (DAC). DACs from shrimp shell and squid pen showed the same degree of digestibility by lysozyme in spite of a difference in the crystal structure of the original chitins. The crystal structure of chitin and the degree of N-acetyl group aggregation among DA-chitin molecules affect the enzymatic digestibility of chitin and DA-chitin, respectively.
Acetylation↗
Ca entry at rest and during prolonged depolarization in dialyzed squid axons.
Ca influx has been studied in squid axons under internal dialysis control. In axons dialyzed with "normal" physiological conditions (Nai = 40-50 mM, Cai2+ = 0.06-0.1 microM, ATP = 2 mM, Ki = 310 mM), 70% of the resting Ca influx is sensitive to external TTX (K0.5 congruent to 5 nM), 20% of it can be accounted by the reversal of the Na-Ca exchange, and the remaining fraction (10%) is insensitive to TTX, D-600, and Nai. The Ca antagonic drug D-600 (50-100 microM) has an inhibitory effect on the resting Ca influx. This compound was found to affect both the TTX sensitive and the Nai-dependent Ca influx components. In the presence of Nai and ATP, Cai2+ activates the carrier mediated Ca entry (Nai-dependent Ca influx). Most of the activation occurs in the submicromolar range of Cai2+ concentrations (K0.5 congruent to 0.6 microM). In the absence of Nai and/or ATP, no activation of Ca influx by Cai2+ was found up to about 5 microM Cai2+. Prolonged depolarization with high Ko causes an increase in Ca influx sustained for long time (minutes). Depolarizing the axons by removing Ki causes the same effect. This depolarization-induced Ca entry was only observed in axons containing Nai. In the absence of Nai, Ca influx decreases with increasing Ko. The activation of the carrier mediated Ca entry (electrogenic Na/Ca exchange) by membrane depolarization was found to be markedly dependent on the magnitude of Ca2+ i. Increasing the magnitude of Ca2+ i from 0.1 to 0.6 microM causes a ten fold increase in the extra Ca influx induced by a K-depolarization.
Animals↗
Calcium clamp of the intracellular environment.
Quantitative analysis of the effects of calcium on cell function requires methods for altering intracellular free Ca in a precise and reproducible manner. Microinjection of Ca is very unreliable largely because of the powerful Ca-binding properties of cytoplasm. Much more satisfactory are microinjection of Ca-buffers - provided enough buffer is introduced - and various forms of intracellular dialysis and perfusion which permit full equilibration of the cell interior with a defined artificial intracellular environment.
Adrenal Medulla↗
Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons.
Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.
Aequorin↗
Calcium entry in squid axons during voltage clamp pulses.
Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.
Aequorin↗
Lipid composition of marine and estuarine invertebrates. Part II: mollusca.
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Animals↗
Design and characterization of electrochromic membrane probes.
Electrochronic membrane probes display a spectroscopic response to membrane potential by a direct electronic mechanism. This allows such probes to be designed a priori via quantum-chemical techniques. The detailed behavior of potentiometric optical probes can be elucidated with an apparatus based on phase-sensitive detection from a hemispherical lipid bilayer; several different types of response spectra can be obtained with this apparatus allowing distinction between the electrochromic mechanism and the more common molecular-motion based mechanisms. The development of 'fast' potentiometric dyes has now reached a stage where practical and exciting applications are rapidly appearing. It is anticipated that the emergence of a complementary set of electrochromic probes will lead to new applications; in particular, it may be possible to elucidate the molecular events which underlie biological or physiological phenomena.
Animals↗
Spatial control of membrane potential: a method for improved voltage clamping of the squid giant synapse.
In voltage clamp experiments at the squid giant synapse anisopotentiality of the presynaptic terminal can distort measurements of synaptic transfer. Local compression of the presynaptic cell, at the point where the terminal joins its axon, appears to reduce this problem by electrically isolating the terminal from the axon. Because compression reduced anisopotentiality in the presynaptic terminal and altered the apparent voltage dependency of transmitter release, this procedure may be a simple way to improve voltage clamp control in this and other long cells. Compression of the presynaptic cell also reduced the diffusion of injected molecules out of the terminal region. This increased the effective concentration of given quantities of tetraethylammonium and Fast green injected into the presynaptic terminal. Thus, compression should also facilitate experiments in which molecules are microinjected into this and other cells because the effective volume of the cell is reduced and less of the injected substance will be required.
Animals↗
Technical aspects of voltage-clamping the cut-open squid giant axon.
The design of a voltage-clamp system dedicated to recording the fluctuation of sodium currents under non-stationary conditions from a leaflet of cut-open squid axon is presented. The membrane leaflet is mechanically sandwiched between the apices of two finely machined plexiglass cones which enable fluid access to each side of the membrane and a known area of membrane to be voltage-clamped. The design requirements necessary to achieve satisfactory signal resolution have been assessed in terms of the overall digitising resolution of the ADC hardware and the intrinsic and extrinsic components of the clamp-system noise. Good agreement between the predicted and measured noise performance was found. The clamp system has enabled simultaneous estimates of the single-channel conductance and channel density to be made over a much wider range of experimental conditions than previously possible.
Animals↗
High resolution recording of asymmetry currents from the squid giant axon: technical aspects of voltage clamp design.
The design, realisation and performance of a voltage clamp system dedicated to recording asymmetry currents from the squid giant axon is presented. The design has been optimised with respect to dynamic response, signal-to-noise ratio and linearity. Analytical expressions are given for both the dynamic performance and noise characteristics which simplify the design and setting up of the clamp and provide excellent agreement between design theory and practice. A 0.5 cm2 area of membrane can be voltage clamped in response to a command input voltage step within 10 microseconds with smooth settling and up to 100% of the effective series resistance being compensated. The noise contributions of the recording chamber, voltage sensing electrodes and clamp electronics have been reduced such that recording of gating currents with a more than 10-fold reduction in the number of averages required compared with previous clamp designs is possible. The overall system nonlinearity results in typically less than 1% contribution to the measured asymmetry charge.
Animals↗
The use of intracellular dialysis to study signal transduction coupling in the squid giant axon.
The squid giant axon has proved a useful model in the study of ionic channel gating, intracellular homeostasis and receptor-mediated signal transduction leading to generation of intracellular second messengers. In the latter category, previous studies on activation of adenylate or guanylate cyclase have used intact and intracellularly perfused axons to investigate the effects of extra- and intracellular agents on the transduction processes. However, the perfusion of the axon interior washes out many factors which may be important in the processes under study. We introduce here the use of porous cellulose dialysis tubing as a means to circumvent these problems. We find that this dialysis technique is a simple procedure to set-up, and the serotonin/G-protein/adenylate cyclase system can readily be studied in the dialysed axon. This approach should allow investigation under conditions which retain asymmetric transmembrane conditions.
Adenylyl Cyclases↗
Optical recording of membrane potential responses from early embryonic chick ganglia using voltage-sensitive dyes.
Changes in absorbance of voltage-sensitive merocyanine-rhodanine dyes were used to monitor electrical responses in the semilunar ganglion of 4-10-day-old developing chick embryos. The electrical responses were simultaneously recorded from many positions in the ganglion. Stimulation of the afferent nerve fibers (the ophthalmic division of cranial nerve V) with a suction electrode led to changes in light absorption of the stained ganglia. With both the depolarizing and hyperpolarizing pulses, the change was largest at 700 nm and was eliminated at a wavelength of 620 nm where the voltage-dependent absorption change of the dyes disappears. In the 4-10-day-old embryonic ganglia, two types of optical membrane potential responses, 'non-conducted' and 'conducted' responses, were identified. The non-conducted response varied with the intensity of the stimulus and had the nature of an electrotonic spread. Furthermore, this non-conducted response exhibited an 'initial upstroke-response' followed by the steady-state plateau evoked by larger depolarizing pulses. The conducted responses were blocked by tetrodotoxin (TTX) or by high external potassium concentration. The incidence of the conducted responses increased as development proceeded from the 5th to the 10th day of age. Thus, the TTX-sensitive action potential activity is probably generated initially in the semilunar ganglion during the 5-10-day-stage of development. These data represent the first demonstration of membrane potential responses in early embryonic intact nervous system. Furthermore, these studies demonstrate the usefulness of voltage-sensitive dyes in the analysis of the organizing process of embryonic neuronal functions during these early stages of development.
Animals↗