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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 11 (1999), S. 0 
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Whole-cell Na+ currents (INa) were recorded in inspiratory neurons in a medullary slice preparation from neonatal mouse that contains the functional respiratory network. Hypoxia and metabolic poisoning with KCN rapidly inhibited INa by reducing the number of Na+ channels available for opening during depolarization. Application of agents specific for G-proteins, protein kinase C and A, intracellular Ca2+ and pH did not prevent the hypoxic inhibition of INa. The effects of hypo-osmolarity and hypoxia were additive, whereas hyperosmolarity partially prevented a subsequent hypoxic inhibition of INa. Cytochalasin B and colchicine decreased, and taxol or phalloidin increased INa and reduced its hypoxic inhibition. We conclude that cytoskeleton rearrangements during hypoxia are responsible for suppression of a fast INa in brainstem respiratory neurons, which could be mediated by the uncoupling of channel inactivation gates from cytoskeletal elements.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 12 (2000), S. 0 
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A hyperpolarization-activated current, Ih, is often implied in pacemaker-like depolarizations during rhythmic oscillatory activity. We describe Ih in the isolated respiratory centre of immature mice (P6–P11). Ih was recorded in 15% (22/146) of all inspiratory neurons examined. The mean half-maximal Ih activation occurred at −78 mV and the reversal potential was −40 mV. Ih was inhibited by Cs+ (1–5 m m) and by organic blockers N-ethyl-1,6-dihydro-1,2-dimethyl-6-(methylimino)-N-phenyl-4-pyrimidinamine (ZD 7288; 0.3–3 μm) and N,N′-bis-(3,4-dimethylphenylethyl)-N-methylamine (YS 035, 3–30 μm), but not by Ba2+ (0.5 m m). The organic Ih blockers did not change the inspiratory bursts recorded from the XIIth nerve and synaptic drives in inspiratory neurons. Hypoxia reversibly inhibited Ih but, in the presence of organic blockers, the hypoxic reaction remained unchanged. We conclude that although Ih channels are functional in a minority of inspiratory neurons, Ih does not contribute to respiratory rhythm generation or its modulation by hypoxia.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 419 (1991), S. 138-143 
    ISSN: 1432-2013
    Keywords: Intracellular pH ; Calcium current ; Lectins ; Weak electrolytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Changes of calcium currents with intracellular pH (pHi) were investigated in chick dorsal root ganglion (DRG) neurones. High-threshold calcium currents decreased after extracellular application of a permeable weak acid, sodium acetate (CH3COONa), and increased when applying a permeable weak base, ammonium chloride (NH4Cl), whereas both compounds were ineffective against the low-threshold calcium current. These weak electrolytes, employed to change pHi, did not alter the kinetic and steady-state parameters of activation and inactivation of the calcium current. Extracellular application of concanavalin A (Con A) and wheat germ agglutinin to elevate pHi increased the high-threshold calcium current. Their effect developed within 2–5 min and was independent of lectin concentration varied from 0.1 to 1 mg/ml. The lectin effects were greatly diminished if Na/H exchange was blocked by amiloride or suppressed by low external sodium. Succinilated Con A and Con A in the presence of d-mannose were less effective. Calcium currents were recorded simultaneously with the pHi, monitored with a proton-sensitive microelectrode. It was found that 50% inhibition of the calcium current occured at pHi of 6.5. Histidine-specific reagents — diethylpyrocarbonate, Rose Bengal and Methylene Blue — prevented the modulation of the calcium conductance by CH3COONa and NH4Cl. Extracellular baclofen and theophylline or intracellular phorbol esters, staurosporine, calmodulin antagonists R24571 and W-13, and neomycine failed to prevent the modulation of the calcium current by weak electrolytes. These observations are consistent with an interaction between intracellular protons and calcium channels.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 424 (1993), S. 183-191 
    ISSN: 1432-2013
    Keywords: Intracellular calcium concentration ; Spatial inhomogeneity ; Ca2+ load and recovery ; Internal Ca2+ stores
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca2+ concentration, [Ca]i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]i. During wash-out [Ca]i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]i was quickly restored. [Ca]i recovery was delayed after treating the cell with 2 μM thapsigargin, an inhibitor of the Ca2+ pump of internal Ca2+ stores. Caffeine (10 mM) transiently increased [Ca]i. A second caffeine application produced smaller [Ca]i changes due to the prior depletion of Ca2+ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 μM) transiently increased [Ca]i both in the standard and Ca2+-free solution. [Ca]i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La3+ and in Ca2+-free solutions, but which was greatly diminished in Na+-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]i transients and in the inward current induction.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 76 (1983), S. 83-93 
    ISSN: 1432-1424
    Keywords: ion selectivity ; calcium channel ; mollusc neurons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The slow inward current carried by Na+ through potential-dependent calcium channels in conditions when divalent cations were removed from the extracellular solution by EDTA has been investigated on isolated internally perfused neurons of the snailHelix pomatia. The calcium channels also acquire the capability to pass monovalent cations if other calcium-binding substances are added to the extracellular solution. Based on these facts the conclusion is made that the immediate reason for the modification of the channel selectivity is the absence of divalent cations in the extracellular medium. All potential-dependent characteristics of the modified calcium channel are shifted by 60 to 70 mV in the hyperpolarizing direction compared with those of the original calcium channel. The series of relative permeabilities for modified calcium channels towards monovalent cations ( $$P_{Na^ + } :P_{Li^ + } :P_{N_2 H_5 ^ + } :P_{NH_3 OH^ + }$$ =1.0∶0.8∶0.55∶0.21) is close to that of common “fast” sodium channels ( $$P_{Na^ + } :P_{Li^ + } :P_{N_2 H_5 ^ + } :P_{NH_3 OH^ + }$$ =1.0∶1.04∶0.44∶0.21). The induced sodium current decreases immediately when the concentration of divalent cations in the extracellular solution is elevated. This decrease is not potential dependent and can be approximated by Langmuir's isotherm with dissociation constants pKCa∶pKSr∶pKBa∶pKMg=6.6∶5.5∶4.8∶4.2. The conclusion is drawn that the calcium channels in the somatic membrane have two ion-selecting filters with different functions —an external one consisting, probably, of several carboxylic groups which bind divalent cations in a highly specific manner and determine the impermeability of the channel to monovalent cations in physiological conditions, and the channel ion-selecting filter including a single carboxylic group normally determining the channel selectivity for different divalent cations.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 141 (1994), S. 231-237 
    ISSN: 1432-1424
    Keywords: Ca2+ channel ; Amidiniums ; Block and permeability ; Selectivity filter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The effect of amidiniums on high-threshold Ca2+ channel currents (I Ca) was studied in chick dorsal root ganglion neurons. Guanidinium reduced I Ca in a dose-dependent fashion. The block was relieved by increasing the concentration of the permeant ions, Ba2+ or Ca2+, suggesting a competition for a common binding site within the channel. Formamidinium and methyl-guanidinium suppressed I Ca with similar potencies, whereas l-arginine had no effect. A neutral amidine, urea, increased I Ca. In Ca2+-free solutions guanidinium and Na+ permeated through the Ca2+ channel equally well. Structure-activity relationship obtained for blocking efficacies of different amidiniums are used to discuss possible configurations of the selectivity filter in the Ca2+ channel.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-1424
    Keywords: snail neurons ; fluorescence measurements ; cytoplasmic Ca ; [Ca] i recovery ; Ca-induced Ca release
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Using the fluorescent probe fura-2, we measured the cytoplasmic concentration of free Ca2+ ([Ca] i ) and its changes in isolated, nonidentified neurons of the snailHelix pomatia. [Ca] i increased during membrane depolarization due to opening of Ca channels in the surface membrane. When the membrane potential returned to the resting level, [Ca] i recovered monoexponentially, with the time constant ranging from 10 to 30 sec. The rate of recovery remained unchanged after treatments that interferred with the normal functioning of both Ca/Na exchange and Ca-ATPase in the surface membrane or mitochondria. [Ca] i recovery slowed down upon cooling according to Q10=2.3 and after intracellular injection of vanadate. The data obtained suggest that the rate of [Ca] i recovery after membrane depolarization is mainly determined by Ca pump of intracellular stores (presumably by the endoplasmic reticulum). Ca release from these stores could be induced in the presence of millimolar caffeine or theophylline in the external medium when [Ca] i increased up to a certain threshold level. This depolarization-induced Ca load triggered further transient increase in [Ca] i , which was accompanied by membrane hyperpolarization due to the development of Ca-activated potassium conductance. 1mm procaine or tetracaine, but not lidocaine, inhibited this Ca-induced Ca release. In some cases stable oscillations of [Ca] i were observed. They could be induced by producing a steady Ca influx by membrane depolarization.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 70 (1982), S. 181-189 
    ISSN: 1432-1424
    Keywords: energy profile ; calcium channel ; mollusc neurons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The current-voltage characteristics of the inward calcium and barium currents at different concentrations of these ions in the extracellular solution have been measured on isolated neurons of the snailHelix pomatia intracellularly perfused with potassium-free solution containing 10mm EDTA. On the basis of these characteristics the energy profile of the calcium channel has been calculated using a model based on the absolute reaction rate theory developed by Eyring. The effect of changes of the near-membrane concentration of the penetrating ions due to the existence of fixed charges on the outer side of the membrane has been taken into account. A satisfactory description of the concentration-and potential-dependence of the calcium inward currents has been obtained based on a three-barrier model for the energy profile of the calcium channel. Calculated dissociation constants for the complexes of Ca2+ ions with the binding sites of the calcium channel have the following values:K out=10mm andK in=2.5mm; and for the complexes of Ba2+ ions,K out=91mm andK in=1.5mm. The outer binding site corresponds to the acidic group with pK=5.8. Comparison between these data and the values of pK for divalent cation complexes with different anionic groups of amino acids allowed us to suggest that the outer binding site contains only one carboxylic group. It was shown that the strength of cation binding to this group determines the conductance of the calcium channel.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 70 (1982), S. 171-179 
    ISSN: 1432-1424
    Keywords: surface potential ; Stern's theory ; mollusc neurons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem. 30∶508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK Ca=90±10,K Sr=60±10,K Ba=25±5 andK Mg=16±5m −1 at pH=7.7 and for groups located in the vicinity of calcium channelsK Ca=67±10,K Sr=20±5 andK Ba=19±5m −1 at pH=7.0. The same groups bind H+ ions with apparent pK=6.2±0.2 that corresponds toK H=1.6×106 m −1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm2 (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm2 (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 86 (1985), S. 197-202 
    ISSN: 1432-1424
    Keywords: surface charge ; mammalian neurones ; microelectrophoresis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M−1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.
    Type of Medium: Electronic Resource
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