Two conformational states involved in the use-dependent TTX blockade of human cardiac Na+ channel

1996 ◽  
Vol 270 (6) ◽  
pp. H2029-H2037 ◽  
Author(s):  
R. Dumaine ◽  
H. A. Hartmann
Keyword(s):  
Direct Interaction ◽  
Na Channel ◽  
Fast Inactivation ◽  
Slow Recovery ◽  
Similar Time ◽  
Activated State ◽  
High Affinity Site ◽  
Action Potential Plateau ◽  
Time Courses ◽  
High Concentrations

We used a fast inactivation-deficient mutant (QQQ) of the human heart Na+ channel alpha-subunit (hH1a) to assess the influence of the inactivation gate on tetrodotoxin (TTX) use-dependent block (UDB) and postrepolarization block (PRB). PRB had similar time courses in both channels, suggesting no direct interaction of the inactivation gate with the TTX binding site. The UDB saturated with high concentrations of TTX in hH1a but not in QQQ, revealing the modulatory action of fast inactivation on UDB. TTX did not stabilize the inactivated states of QQQ, and the extra block developing during long depolarizations suggests a higher-affinity site involved in the gating of the channel. These results cannot be solely explained by a slow recovery from the block in the inactivated states. They suggest a common use-dependent block mechanism for hH1a and QQQ involving a high-affinity site. We propose that an activated state is primarily responsible for UDB during short depolarization in the range of the action potential plateau and that fast inactivation modulates the accessibility of the toxin to this site.

Ryanodine-Sensitive Stores Regulate the Excitability of AH Neurons in the Myenteric Plexus of Guinea-Pig Ileum

2000 ◽  
Vol 84 (6) ◽  
pp. 2777-2785 ◽  
Author(s):  
K. Hillsley ◽  
J. L. Kenyon ◽  
T. K. Smith
Keyword(s):  
Sensory Processing ◽  
Calcium Release ◽  
Excitatory Postsynaptic Potential ◽  
Similar Time ◽  
Intracellular Ca ◽  
Time Courses ◽  
High Concentrations ◽  
Calcium Induced Calcium Release ◽  
Activity Dependent

Myenteric afterhyperpolarizing (AH) neurons are primary afferent neurons within the gastrointestinal tract. Stimulation of the intestinal mucosa evokes action potentials (AP) that are followed by a slow afterhyperpolarization (AHPslow) in the soma. The role of intracellular Ca2+ ([Ca2+]i) and ryanodine-sensitive Ca2+ stores in modulating the electrical activity of myenteric AH neurons was investigated by recording membrane potential and bis-fura-2 fluorescence from 34 AH neurons. Mean resting [Ca2+]i was ∼200 nM. Depolarizing current pulses that elicited APs evoked AHPslow and an increase in [Ca2+]i, with similar time courses. The amplitudes and durations of AHPslow and the Ca2+ transient were proportional to the number of evoked APs, with each AP increasing [Ca2+]i by ∼50 nM. Ryanodine (10 μM) significantly reduced both the amplitude and duration (by 60%) of the evoked Ca2+ transient and AHPslow over the range of APs tested (1–15). Calcium-induced calcium release (CICR) was graded and proportional to the number of APs, with each AP triggering a rise in [Ca2+]i of ∼30 nM Ca2+ via CICR. This indicates that CICR amplifies Ca2+ influx. Similar changes in [Ca2+]i and AHPslow were evoked by two APs in control and six APs in ryanodine. Thus, the magnitude of the change in bulk [Ca2+]i and not the source of the Ca2+ is the determinant of the magnitude of AHPslow. Furthermore, lowering of free [Ca2+]i, either by reducing extracellular Ca2+ or injecting high concentrations of Ca2+buffer, induced depolarization, increased excitability, and abolition of AHPslow. In addition, activation of synaptic input to AH neurons elicited a slow excitatory postsynaptic potential (sEPSP) that was completely blocked in ryanodine. These results demonstrate the importance of [Ca2+]i and CICR in sensory processing in AH neurons. Activity-dependent CICR may be a mechanism to grade the output of AH neurons according to the intensity of sensory input.

scholarly journals BTX modification of Na channels in squid axons. I. State dependence of BTX action.

1991 ◽  
Vol 97 (3) ◽  
pp. 499-519 ◽  
Author(s):  
J Tanguy ◽  
J Z Yeh
Keyword(s):  
Time Constant ◽  
Slow Rate ◽  
Open Channel ◽  
Voltage Dependence ◽  
State Dependence ◽  
Na Channel ◽  
Na Channels ◽  
Fast Inactivation ◽  
Similar Time ◽  
Chloramine T

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.

scholarly journals Fast and slow steps in the activation of sodium channels.

1979 ◽  
Vol 74 (6) ◽  
pp. 691-711 ◽  
Author(s):  
C M Armstrong ◽  
W F Gilly
Keyword(s):  
Early Phase ◽  
Slow Component ◽  
Na Channel ◽  
Gating Current ◽  
Similar Time ◽  
Voltage Range ◽  
Subsequent Step ◽  
Time Courses ◽  
Kinetics Of ◽  
Kinetic Features

Kinetic features of sodium conductance (gNa) and associated gating current (Ig) were studied in voltage-clamped, internally perfused squid axons. Following a step depolarization Ig ON has several kinetic components: (a) a rapid, early phase largely preceding gNa turn-on; (b) a delayed intermediate component developing as gNa increases; and (c) a slow component continuing after gNa is fully activated. With small depolarizations the early phase shows a quick rise (less than 40 mus) and smooth decay; the slow component is not detectable. During large pulses all three components are present, and the earliest shows a rising phase or initial plateau lasting approximately 80 mus. Steady-state and kinetic features of Ig are minimally influenced by control pulse currents, provided controls are restricted to a sufficiently negative voltage range. Ig OFF following a strong brief pulse also shows a rising phase. A depolarizing prepulse producing gNa inactivation and Ig immobilization eliminates the rising phase of Ig OFF. gNa, the immobilized portion of Ig ON, and the rising phase reappear with similar time-courses when tested with a second depolarizing pulse after varying periods of repolarization. 30 mM external ZnCl2 delays and slows gNa activation, prolongs the rising phase, and slows the subsequent decay of Ig ON. Zn does not affect the kinetics of gNa tails or Ig OFF as channels close, however. We present a sequential kinetic model of Na channel activation, which adequately describes the observations. The rapid early phase of IgON is generated by a series of several fast steps, while the intermediate component reflects a subsequent step. The slow component is too slow to be clearly associated with gNa activation.

scholarly journals Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential.

1992 ◽  
Vol 99 (3) ◽  
pp. 317-338 ◽  
Author(s):  
L Reuss ◽  
B Simon ◽  
C U Cotton
Keyword(s):  
Time Course ◽  
Bathing Solution ◽  
Intercellular Spaces ◽  
Similar Time ◽  
Streaming Potentials ◽  
Osmotic Water ◽  
Lateral Intercellular Spaces ◽  
Transepithelial Voltage ◽  
Time Courses ◽  
Good Agreement

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.

scholarly journals Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase

1991 ◽  
Vol 279 (2) ◽  
pp. 343-350 ◽  
Author(s):  
J Nari ◽  
G Noat ◽  
J Ricard
Keyword(s):  
Cell Wall ◽  
Metal Ions ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Direct Interaction ◽  
Enzyme Reaction ◽  
Pectin Methylesterase ◽  
High Concentrations ◽  
Enzyme Molecules ◽  
Hydrolysis Of

The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur. The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme. With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase. This is explained by the existence of ‘blocks’ of carboxy groups on pectin that may trap enzyme molecules and thus prevent the enzyme reaction occurring. Metal ions may interact with these negatively charged groups, thus allowing the enzyme to interact with the ester bonds to be cleaved. At high concentrations, however, metal ions inhibit the enzyme reaction. This is again understandable on the basis of the view that some carboxy groups must be adjacent to the ester bond to be cleaved in order to allow the reaction to proceed. Indeed, if these groups are blocked by metal ions, the enzyme reaction cannot occur, and this is the reason for the apparent inhibition of the reaction by high concentrations of metal ions. Methylene Blue, which may be bound to pectin, may replace metal ions in the ‘activation’ and ‘inhibition’ of the enzyme reaction. A kinetic model based on these results has been proposed and fits the kinetic data very well. All the available results favour the view that metal ions do not affect the reaction through a direct interaction with enzyme, but rather with pectin.

scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

The effects of ethanol (0.75 g/kg body weight) on the activities of selected enzymes in sera of healthy young adults: 1. Intermediate-term effects.

1977 ◽  
Vol 23 (5) ◽  
pp. 830-834 ◽  
Author(s):  
D E Freer ◽  
B E Statland
Keyword(s):  
Young Adults ◽  
Body Weight ◽  
Enzyme Activity ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Ethanol Ingestion ◽  
Similar Time ◽  
Gamma Glutamyltransferase ◽  
Time Courses ◽  
Apparently Healthy

Abstract We report the intermediate-term effects of three consecutive evenings of moderate ethanol ingestion (0.75 g/kg body weight each evening) on activity values for alkaline phosphatase, gamma-glutamyltransferase, creatine kinase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in sera of nine apparently healthy young adults. We define "intermediate-term" effects as those occurring between 10 h and 100 h after completion of the ethanol consumption schedule. The most pronounced changes in enzyme activity for the group of volunteers were: gamma-glutamyltransferase, +25% at 60 h after ethanol ingestion; alanine aminotransferase, +12% at 60 h after ethanol; and aspartate aminotransferase,--12% at 60 h after ethanol. All three enzymes exhibited similar time courses, i.e., mean peak activity changes were observed at 60 h, and all three mean enzyme activity values returned to near baseline by 100 h. The possible explanations for the observed changes and the clinical significance are discussed.

Serine proteases increase oxidative stress in lung cells

2001 ◽  
Vol 281 (3) ◽  
pp. L556-L564 ◽  
Author(s):  
Kazutetsu Aoshiba ◽  
Kimihiko Yasuda ◽  
Shuji Yasui ◽  
Jun Tamaoki ◽  
Atsushi Nagai
Keyword(s):  
Oxidative Stress ◽  
Serine Proteases ◽  
Direct Interaction ◽  
Lung Fibroblasts ◽  
Lung Cells ◽  
Cellular Injury ◽  
Oxygen Species ◽  
Cytotoxic Effects ◽  
Bronchial Epithelial ◽  
High Concentrations

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1–100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2′-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H2O2, and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.

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