The Experts below are selected from a list of 168 Experts worldwide ranked by ideXlab platform
F Bezanilla - One of the best experts on this subject based on the ideXlab platform.
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single channel macroscopic and gating currents from sodium channels in the Squid Giant Axon
Biophysical Journal, 1991Co-Authors: Carol A Vandenberg, F BezanillaAbstract:Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open Squid Giant Axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9–13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.
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a sodium channel gating model based on single channel macroscopic ionic and gating currents in the Squid Giant Axon
Biophysical Journal, 1991Co-Authors: Carol A Vandenberg, F BezanillaAbstract:Sodium channel gating behavior was modeled with Markovian models fitted to currents from the cut-open Squid Giant Axon in the absence of divalent cations. Optimum models were selected with maximum likelihood criteria using single-channel data, then models were refined and extended by simultaneous fitting of macroscopic ionic currents, ON and OFF gating currents, and single-channel first latency densities over a wide voltage range. Best models have five closed states before channel opening, with inactivation from at least one closed state as well as the open state. Forward activation rate constants increase with depolarization, and deactivation rate constants increase with hyperpolarization. Rates of inactivation from the open or closed states are generally slower than activation or deactivation rates and show little or no voltage dependence. Channels tend to reopen several times before inactivating. Macroscopic rates of activation and inactivation result from a combination of closed, open and inactivated state transitions. At negative potentials the time to first opening dominates the macroscopic current due to slow activation rates compared with deactivation rates: channels tend to reopen rarely, and often inactivate from closed states before they reopen. At more positive potentials, the time to first opening and burst duration together produce the macroscopic current.
Francisco Bezanilla - One of the best experts on this subject based on the ideXlab platform.
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gating kinetics of batrachotoxin modified na channels in the Squid Giant Axon voltage and temperature effects
Biophysical Journal, 1992Co-Authors: Ana M Correa, Francisco Bezanilla, Ramon LatorreAbstract: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.
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phosphorylation of k channels in the Squid Giant Axon a mechanistic analysis
Journal of Bioenergetics and Biomembranes, 1991Co-Authors: Eduardo Perozo, Francisco BezanillaAbstract:Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In Squid Giant Axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.
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single channel studies of the phosphorylation of k channels in the Squid Giant Axon ii nonstationary conditions
The Journal of General Physiology, 1991Co-Authors: Eduardo Perozo, Deshien Jong, Francisco BezanillaAbstract:The effects of phosphorylation on the properties of the 20-pS channel of the Squid Giant Axon were studied using the cut-open Axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed Axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.
Carol A Vandenberg - One of the best experts on this subject based on the ideXlab platform.
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single channel macroscopic and gating currents from sodium channels in the Squid Giant Axon
Biophysical Journal, 1991Co-Authors: Carol A Vandenberg, F BezanillaAbstract:Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open Squid Giant Axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9–13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.
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a sodium channel gating model based on single channel macroscopic ionic and gating currents in the Squid Giant Axon
Biophysical Journal, 1991Co-Authors: Carol A Vandenberg, F BezanillaAbstract:Sodium channel gating behavior was modeled with Markovian models fitted to currents from the cut-open Squid Giant Axon in the absence of divalent cations. Optimum models were selected with maximum likelihood criteria using single-channel data, then models were refined and extended by simultaneous fitting of macroscopic ionic currents, ON and OFF gating currents, and single-channel first latency densities over a wide voltage range. Best models have five closed states before channel opening, with inactivation from at least one closed state as well as the open state. Forward activation rate constants increase with depolarization, and deactivation rate constants increase with hyperpolarization. Rates of inactivation from the open or closed states are generally slower than activation or deactivation rates and show little or no voltage dependence. Channels tend to reopen several times before inactivating. Macroscopic rates of activation and inactivation result from a combination of closed, open and inactivated state transitions. At negative potentials the time to first opening dominates the macroscopic current due to slow activation rates compared with deactivation rates: channels tend to reopen rarely, and often inactivate from closed states before they reopen. At more positive potentials, the time to first opening and burst duration together produce the macroscopic current.
Thomas S. Reese - One of the best experts on this subject based on the ideXlab platform.
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kinesin 3 is an organelle motor in the Squid Giant Axon
Traffic, 2008Co-Authors: Thomas S. Reese, Joseph A Degiorgis, Tatyana A Petukhova, Teresa A EvansAbstract:Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the Squid Giant Axon, where it is thought to move organelles on microtubules. In this study, we identify a second Squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the Axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the Axon for unidirectional movement of organelles along microtubules.
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slow transport of unpolymerized tubulin and polymerized neurofilament in the Squid Giant Axon
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: James A. Galbraith, Paul E. Gallant, Thomas S. Reese, Michelle L SchliefAbstract:A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in Axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the Squid Giant Axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm/day, whereas neurofilament was moved at 1.1 mm/day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the Axon with cold in the presence of nocodazole reduced transport of both proteins to ≈20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 μm2/s compared with only 0.43 μm2/s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole/cold-sensitive transport mechanism.
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organization of the cortical endoplasmic reticulum in the Squid Giant Axon
Journal of Neurocytology, 1997Co-Authors: J Metuzals, K Hammar, D Chang, Thomas S. ReeseAbstract:The organization of the cortical endoplasmic reticulum in the Squid Giant Axon was investigated by rapid freeze and freeze-substitution electron microscopy, thereby eliminating the effects of fixatives on this potentially labile structure. Juvenile Squid, which have thinner Schwann sheaths, were used in order to achieve freezing deep enough to include the entire Axonal cortex. The smooth endoplasmic reticulum is composed of subaxolemmal and deeper cisternae, tubules, tethers and vesicles. The subaxolemmal cisternae make junctional contacts with the axolemma which are characterized by filamentous-granular bridging structures approximately 3 nm in diameter. The subaxolemmal junctions with the axolemma resemble the coupling junctions between the sarcoplasmic reticulum and the T-tubules in muscle. Reconstruction of short series of sections showed that a number of the elements of the endoplasmic reticulum were continuous but numerous separate vesicles were present as well. The morphology of endoplasmic reticulum as described here suggests that it is a highly dynamic entity as well as a Ca2+ sequestering organelle.
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Transport of cytoskeletal elements in the Squid Giant Axon.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Mark Terasaki, Paul E. Gallant, Alexandra Schmidek, James A. Galbraith, Thomas S. ReeseAbstract:In order to explore how cytoskeletal proteins are moved by Axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into Squid Giant Axons and observed their movements by confocal microscopy. The Squid Giant Axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the Axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the Axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the Axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the Axon.
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cytoplasmic constriction and vesiculation after axotomy in the Squid Giant Axon
Journal of Neurocytology, 1995Co-Authors: Paul E. Gallant, K Hammar, Thomas S. ReeseAbstract:The Squid Giant Axon responded to a transection injury by producing a gradient of cytoplasmic and vesicular changes at the cut end. At the immediate opening of the cut Axon the cytoplasm was fragmented and dispersed and the vesicles in this region were in rapid Brownian movement. Approximately 0.1 mm further in, at the site of maximal Axonal constriction, the axoplasm was condensed into a compact, constricted mass containing many large vesicles. The axoplasm was normal a few millimetres beyond this constricted, vesiculated end. It appears that transection triggered the transformation of normal axoplasm into a tightly constricted, highly vesiculated structure. This modified axoplasm at the cut end may slow the spread of damage and degeneration by preventing the bulk outflow of axoplasm, by slowing down the loss of intracellular molecules and by slowing down the influx of destructive extracellular ions (like calcium and chloride).
Richard Darwin Keynes - One of the best experts on this subject based on the ideXlab platform.
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on the slowly rising phase of the sodium gating current in the Squid Giant Axon
Proceedings of The Royal Society B: Biological Sciences, 1998Co-Authors: Richard Darwin Keynes, Fredrik ElinderAbstract:High-resolution records of the sodium gating current in the Squid Giant Axon demonstrate the existence of a slowly rising phase that is first apparent at pulse potentials slightly below zero, and becomes increasingly pronounced at more positive potentials. At +80 mV the current reaches its peak with a delay of 30 microseconds at 10 degrees C. It is suggested that this current is generated by the first two steps labelled R-->P and P-->A in the S4 units of all four domains of the series-parallel gating system, activating the channel before its opening by the third steps A-->B in domains I, II and III in conjunction with hydration. The kinetics of the slowly rising phase can only be explained by the incorporation of an appropriate degree of voltage-dependent cooperativity between the S4 voltage-sensors for their two initial transitions.
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blocking of the sodium channel by external tris in the Squid Giant Axon
Proceedings of The Royal Society B: Biological Sciences, 1991Co-Authors: Richard Darwin Keynes, Eduardo Rojas, Valentin CenaAbstract:The macroscopic current carried by Na + ions was recorded in voltage-clamped Squid Giant Axons that were dialysed with a mixture of CsF and NaF, and bathed in chloride solutions in which the sodium current (1 Na ) was reduced by partly blocking the sodium channels with tetrodotoxin, or by replacing four fifths of the Na + either with choline or with tris buffer at pH 7.3. The permeability coefficient (TNa fast) for the fast-inactivating current was unchanged on substitution of choline for sodium in the bathing solution, but in the high-Tris bathing solution, TNa fagt was reduced in a voltage-dependent manner, so that the permeability relative to normal rose from 0.2 at —40 m V to 0.8 at +100 mV. The coefficie10t TNa non for the non-inactivating current behaved in a similar way, except that the permeability ratio fell between test potentials of —40 and + 10 mV instead of rising. The blocking effect of Tris was unaffected by temperature, but there was some interaction with that of internal tetramethylammonium ions.
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On the voltage dependence of inactivation in the sodium channel of the Squid Giant Axon.
Proceedings. Biological sciences, 1991Co-Authors: Richard Darwin KeynesAbstract:Measurements of the macroscopic sodium current in the Squid Giant Axon show that the inactivation gate carries around 1.3 units of electronic charge. The contrary evidence from single-channel studies is considered, and a modified series-parallel model of the sodium channel is proposed that might help to resolve the disagreement.