The Experts below are selected from a list of 195 Experts worldwide ranked by ideXlab platform
Francisco Bezanilla - One of the best experts on this subject based on the ideXlab platform.
-
Phosphorylation Modulates Potassium Conductance and Gating Current of Perfused Giant Axons of Squid
2016Co-Authors: F. Phosphorylation, Francisco Bezanilla, Christina K. AugustineAbstract:modulates potassium conductance and gating current of perfused Giant Axon of squid. J. Gen
-
k translocation by the Giant Axon of the humboldt squid na k atpase
Biophysical Journal, 2014Co-Authors: Juan P Castillo, Francisco Bezanilla, Ramon Latorre, Daniel Basilio, Miguel HolmgrenAbstract:The Na+/K+ pump is a membrane protein which plays a fundamental role in maintaining the Na+ and K+ electrochemical gradients in animal cells. When internal and external Na+ is absent the pump can only undergo K+ translocation reactions. At equilibrium, the distribution of the different protein conformations depends on the rate constants of each step leading to K+ binding and unbinding. If some of these rate constants are voltage-dependent, sudden changes in membrane electric potential will shift the binding-unbinding equilibrium. In those translocation reactions, K+ has to travel a fraction of the membrane electric field generating a transient current signal. Here, K+ pump currents were measured under voltage clamp conditions using the Giant Axon of the Humboldt squid, which due to its large diameter (1 -1.5 mm) allows the detection of these charge movements. By using H2DTG, a reversible inhibitor of the squid Na+/K+ pump, we were able to obtain H2DTG-sensitive transient currents in response to voltage jumps in K+/K+ conditions. Kinetics of these transient currents shows two main components, that in contrast to their Na+ counterpart, appeared to be uncoupled. The origin of the fast component appears to be the movement of ions along an access channel that it is always open, suggesting that the gate that occlude K ions is deep in the permeation pathway. On the other hand, charge displacement distribution and rate constants of the slow component show a clear dependence on the K+ external concentration revealing that the entrance of the K+ to the Na+/K+ pump from the external side is a voltage-dependent step. Supported by FIRCA grant R03 TW008351 and U54GM087519, GM030376, NS64259, HL36783 and the Intramural Program of the NINDS/NIH and FONDECYT 1110430.
-
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.
-
single channel macroscopic and gating currents from sodium channels in the squid Giant Axon
Biophysical Journal, 1991Co-Authors: Carol A Vandenberg, Francisco 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.
-
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, Francisco 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.
Carol A Vandenberg - One of the best experts on this subject based on the ideXlab platform.
-
single channel macroscopic and gating currents from sodium channels in the squid Giant Axon
Biophysical Journal, 1991Co-Authors: Carol A Vandenberg, Francisco 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.
-
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, Francisco 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.
Harish C. Pant - One of the best experts on this subject based on the ideXlab platform.
-
Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.
Cellular and molecular neurobiology, 2016Co-Authors: Harold Gainer, Shirley B. House, Dong Sun Kim, Hemin Chin, Harish C. PantAbstract: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.
-
characterization of the phosphorylation sites of the squid loligo pealei high molecular weight neurofilament protein from Giant Axon axoplasm
Journal of Neurochemistry, 2001Co-Authors: Howard Jaffe, Pushkar Sharma, Philip Grant, Harish C. PantAbstract:Axonal caliber in vertebrates is attributed, in part, to the extensive phosphorylation of NFM and NFH C-terminal tail domain KSP repeats by proline-directed kinases. The squid Giant Axon, primarily involved in rapid impulse conduction during jet propulsion motility, is enriched in squid-specific neurofilaments, particularly the highly phosphorylated NF-220. Of the 228 serine-threonine candidate phosphate acceptor sites in the NF-220 tail domain (residues 401–1220), 82 are found in numerous repeats of three different motifs SAR/K, SEK/R, K/RSP, with 62 of these tightly clustered in the C-terminal repeat segment (residues 840–1160). Characterization of the in vivo NF-220 phosphorylated sites should provide clues as to the relevant kinases. To characterize these sites, proteolytic digests of NF-220 were analyzed by a combination of HPLC, electrospray tandem mass spectrometry and database searching. A total of 53 phosphorylation sites were characterized, with 47 clustered in the C-terminal repeat segment (residues 840–1160), representing 76% (47/62) of the total acceptor sites in the region. As in mammalian NFH, approximately 64% of the K/RSP sites (14/22) in this region were found to be phosphorylated implicating proline-directed kinases. Significantly, 78% of serines (31/40) in the KAES*EK and EKS*ARSP motifs were also phosphorylated suggesting that non proline-directed kinases such as CKI may also be involved. This is consistent with previous studies showing that CKI is the principal kinase associated with axoplasmic NF preparations. It also suggests that phosphorylation of large macromolecules with multiple phospho-sites requires sequential phosphorylation by several kinases.
Mayumi Takasaki - One of the best experts on this subject based on the ideXlab platform.
-
dimethylsulfoxide potentiates the nerve conduction blocking effect of lidocaine without augmentation of the intracellular lidocaine concentration in the Giant Axon of crayfish in vitro
Fundamental & Clinical Pharmacology, 2013Co-Authors: Takeshi Yano, Shoichiro Ibusuki, Mayumi Takasaki, Isao TsuneyoshiAbstract:The purpose of this study was to investigate how dimethylsulfoxide (DMSO) potentiates the blocking action of lidocaine. A Giant Axon removed from a crayfish was used to investigate nerve conduction and intracellular lidocaine concentration. The maximum values of the differential waveform (dV/dt max) calculated from evoked action potentials were used for evaluating an inhibition of nerve conduction. The inhibition of the dV/dt max in low-frequency stimulation (tonic block) and high-frequency stimulation (phasic block) after perfusion of 1 mm lidocaine with or without 0.2 vol % DMSO, in which the concentration of DMSO alone had no anesthetic effect, was measured to evaluate the potentiating action of DMSO. The intracellular lidocaine concentration was measured via a lidocaine-sensitive glass microelectrode during 30 min of perfusion of 1 mm lidocaine alone or in combination with DMSO. When applied without lidocaine, DMSO caused a dose-dependent nerve conduction block when used at concentrations >1 vol %. The dV/dt max in the tonic block was significantly decreased when 0.2 vol % DMSO was added to the lidocaine solution (P = 0.004). In the phasic block, there was no significant potentiating action of DMSO. There were no significant differences in the intracellular lidocaine concentrations with or without DMSO. The potentiating effects of DMSO were observed only in the condition of low-frequency stimulation and were not related to the intracellular lidocaine concentration in the Giant Axon of crayfish in vitro.
-
a comparison of intracellular lidocaine and bupivacaine concentrations producing nerve conduction block in the Giant Axon of crayfish in vitro
Anesthesia & Analgesia, 2006Co-Authors: Takeshi Yano, Shoichiro Ibusuki, Mayumi TakasakiAbstract:Clinically, lidocaine requires a larger concentration than bupivacaine to block nerves. Bupivacaine has a higher lipid solubility, tissue permeability, and affinity for sodium channels than lidocaine, resulting in greater anesthetic potency. Local anesthetics require access to the sodium channel from the intracellular milieu. In this study, we sought to determine the intracellular concentration of lidocaine and bupivacaine when a nerve was blocked in the Giant Axon of a crayfish. A solution of lidocaine or bupivacaine was perfused, and a nerve block was determined as the absence of an evoked action potential after tonic or phasic electrical stimulation. The intracellular lidocaine or bupivacaine concentration was measured using a lidocaine- or bupivacaine-sensitive glass micro-electrode. A phasic block was more effectively and rapidly achieved with a smaller concentration of bupivacaine than with lidocaine. The intracellular concentration and intra- to extracellular ratios were significantly larger with lidocaine than with bupivacaine when nerve conduction was blocked. These findings suggest that bupivacaine has a higher potency than lidocaine, at least in the Giant Axon of a crayfish in vitro. The implications of the present results are that bupivacaine is a more potent nerve block and produces a use-dependent (phasic) block at smaller concentrations than lidocaine.
-
comparisons of the anesthetic potency and intracellular concentrations of s and r bupivacaine and ropivacaine in crayfish Giant Axon in vitro
Anesthesia & Analgesia, 2000Co-Authors: Yuko Kanai, Hiroshi Katsuki, Mayumi TakasakiAbstract:UNLABELLED Levobupivacaine and ropivacaine are both single S(-) enantiomers that have less severe cardiotoxic and convulsant effects than racemic bupivacaine. We compared the anesthetic actions of S(-) bupivacaine, R(+) bupivacaine, and ropivacaine in vitro by studying their effects on action potential amplitude and the maximal rate of rise of action potential in crayfish Giant Axon. To clarify the difference of intracellular anesthetic concentration, the intracellular ionized anesthetic concentration was measured. Desheathed crayfish Axons were stimulated at a frequency of either 0. 1 or 5 Hz and perfused with 1 mM of each anesthetic at pH 7.0. Intracellular anesthetic concentration was measured by us- ing local anesthetic-sensitive glass microelectrodes. At 0.1-Hz stimulation, no differences were observed in their potency. At 5-Hz stimulation, the order of magnitude of the mean percentage decrease in maximal rate of rise of action potential was S(-) bupivacaine > R(+) bupivacaine > ropivacaine. Intracellular local anesthetic concentration did not differ among the three anesthetics at 0.1 Hz and 5 Hz. We conclude that, compared with ropivacaine, S(-) bupivacaine has a more potent phasic blocking effect in crayfish Giant Axon. The intracellular local anesthetic concentrations of S(-), R(+) bupivacaine and ropivacaine were not significantly different, regardless of differences in blocking effect and stimulation frequency. IMPLICATIONS S(-) bupivacaine has a more potent phasic blocking effect than ropivacaine or R(+) bupivacaine in crayfish Giant Axons in vitro. An equivalent intracellular local anesthetic concentration for the three anesthetics was found, suggesting that the intracellular cationic local anesthetic concentration is not directly correlated with the intensity of block.
Thomas S. Reese - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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.
-
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.
-
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.
-
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).