scholarly journals Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

1998 ◽  
Vol 112 (1) ◽  
pp. 85-93 ◽  
Author(s):  
John G. Starkus ◽  
Lioba Kuschel ◽  
Martin D. Rayner ◽  
Stefan H. Heinemann
Keyword(s):  
Structural Changes ◽  
Ionic Current ◽  
Outer Region ◽  
Ionic Currents ◽  
Shaker Potassium Channel ◽  
Shaker Channels ◽  
Gating Charge ◽  
Na Currents ◽  
Shaker Potassium Channels ◽  
Kinetics Of

C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.

scholarly journals Shaker potassium channel gating. I: Transitions near the open state.

1994 ◽  
Vol 103 (2) ◽  
pp. 249-278 ◽  
Author(s):  
T Hoshi ◽  
W N Zagotta ◽  
R W Aldrich
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Activation Process ◽  
Time Data ◽  
Shaker Potassium Channel ◽  
Voltage Dependent ◽  
Average Current ◽  
Shaker Potassium Channels ◽  
Kinetics Of ◽  
Deactivation Process

Kinetics of single voltage-dependent Shaker potassium channels expressed in Xenopus oocytes were studied in the absence of fast N-type inactivation. Comparison of the single-channel first latency distribution and the time course of the ensemble average current showed that the activation time course and its voltage dependence are largely determined by the transitions before first opening. The open dwell time data are consistent with a single kinetically distinguishable open state. Once the channel opens, it can enter at least two closed states which are not traversed frequently during the activation process. The rate constants for the transitions among these closed states and the open state are nearly voltage-independent at depolarized voltages (> -30 mV). During the deactivation process at more negative voltages, the channel can close directly to a closed state in the activation pathway in a voltage-dependent fashion.

scholarly journals Investigating the Putative Glycine Hinge in Shaker Potassium Channel

2005 ◽  
Vol 126 (3) ◽  
pp. 213-226 ◽  
Author(s):  
Shinghua Ding ◽  
Lindsey Ingleby ◽  
Christopher A. Ahern ◽  
Richard Horn
Keyword(s):  
Potassium Channel ◽  
Functional Expression ◽  
Shaker Potassium Channel ◽  
Glycine Residue ◽  
Shaker Channels ◽  
Channel Opening ◽  
Activation Gating ◽  
Voltage Dependent ◽  
Shaker Potassium Channels

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523–526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

Desarrollo de un simulador de la corriente funny del nodo sinusal para la enseñanza-aprendizaje

2021 ◽  
Vol 2 (5) ◽  
pp. 6682-6693
Author(s):  
Jessica Quintero Pérez ◽  
Arturo Reyes Lazalde ◽  
Rosa María Reyes Chapero ◽  
Marleni Reyes Monreal ◽  
María Eugenia Pérez Bonilla
Keyword(s):  
Sinus Node ◽  
Ionic Current ◽  
Ionic Currents ◽  
Visual Basic ◽  
Pacemaker Activity ◽  
Basic Language ◽  
Diastolic Depolarization ◽  
Didactic Material ◽  
Visual Basic Language ◽  
Kinetics Of

Las células del nodo sinusal generan la principal actividad eléctrica marcapaso del corazón. La actividad marcapaso se debe a la presencia de la corriente funny (If). Esta corriente iónica es entrante y se activa con la hiperpolarización en rangos de voltaje presentes durante la fase de despolarización diastólica; contrario a la mayoría de las corrientes iónicas que se activan con la despolarización. El canal funny es permeable a iones Na+ y K+. Generalmente, en el curso de biofísica a nivel de licenciatura los alumnos conocen algunos canales dependientes de voltaje del tipo de Hodgkin y Huxley que se activan con la despolarización. Sin embargo, no se estudia ningún canal que se active con una hiperpolarización. En este trabajo se diseñó y desarrolló un simulador para el estudio y comprensión de la corriente funny presente en el nodo sinusal del conejo. El simulador fue programado en lenguaje Visual Basic ver. 6.0 para ambiente Windows® de XP a Windows® 10. El usuario puede realizar los experimentos con la técnica de fijación de voltaje, modificar las variables y concentraciones de Na+ y K+ externos y observar el efecto en la amplitud de If y la cinética de la curva I-V. Se recomienda su uso como material didáctico de apoyo durante los cursos de biofísica y fisiología en una sala de cómputo o a distancia en cualquier computadora personal con recursos mínimos.   The sinus node cells generate the main electric pacemaker activity of the heart. The pacemaker activity is due to the presence of the current funny (If). This ionic current is incoming and is activated with hypolarization in voltage ranges present during the diastolic depolarization phase, contrary to most ionic currents that are activated by depolarization. The funny channel is permeable to Na+ and K+ ions. Generally, students enrolled in the biophysics course at the undergraduate level are familiar with some voltage-gated channels of the Hodgkin and Huxley type. However, no channel that is activated by hiperpolarization is studied. In this work, a simulator was designed and developed for the study and understanding of the funny current present in the sinus node of rabbits. The simulator was programmed in Visual Basic Language ver. 6.0 for Windows® environment from XP to Windows® 10. The user can perform the experiments with the voltage clamp technique, modify the variables and concentrations of external Na+ and K+ and observe the effect on the amplitude of If and the kinetics of the curve I-V. It is recommended to be used as a support didactic material during biophysics and physiology courses in a computer room or remotely on any personal computer with minimal resources.

scholarly journals Fast Inactivation in Shaker K+ Channels

1998 ◽  
Vol 111 (5) ◽  
pp. 625-638 ◽  
Author(s):  
Michel J. Roux ◽  
Riccardo Olcese ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Fast Inactivation ◽  
Gating Current ◽  
Gating Currents ◽  
Current Decay ◽  
Gating Charge ◽  
External Potassium ◽  
Kinetics Of ◽  
Full Charge

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals Gating and Ionic Currents Reveal How the BKCa Channel's Ca2+ Sensitivity Is Enhanced by its β1 Subunit

2005 ◽  
Vol 126 (4) ◽  
pp. 393-412 ◽  
Author(s):  
Lin Bao ◽  
Daniel H. Cox
Keyword(s):  
Smooth Muscle ◽  
Muscle Tone ◽  
Ionic Current ◽  
Ionic Currents ◽  
Voltage Sensor ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Specific Expression ◽  
Gating Charge ◽  
Sensor Activation

Large-conductance Ca2+-activated K+ channels (BKCa channels) are regulated by the tissue-specific expression of auxiliary β subunits. β1 is predominately expressed in smooth muscle, where it greatly enhances the BKCa channel's Ca2+ sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The β1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BKCa channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with β1 present. Furthermore, β1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by ∼24% (16.8→12.8). The effects of β1 on voltage sensing enhance the BKCa channel's Ca2+ sensitivity by decreasing at most voltages the work that Ca2+ binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca2+ affinity brought about by β1 at negative voltages, our studies suggest that β1 also decreases the true Ca2+ affinity of the closed channel, increasing its Ca2+ dissociation constant from ∼3.7 μM to between 4.7 and 7.1 μM, depending on how many binding sites are affected.

Gating kinetics of ShakerK+ channels are differentially modified by general anesthetics

1998 ◽  
Vol 275 (4) ◽  
pp. C1009-C1021 ◽  
Author(s):  
Ana M. Correa
Keyword(s):  
Xenopus Oocytes ◽  
Ionic Currents ◽  
General Anesthetics ◽  
Gating Currents ◽  
Voltage Gated ◽  
Gating Kinetics ◽  
Shaker Channels ◽  
Gated Channel ◽  
Sites Of Action ◽  
Kinetics Of

The ShakerB K+ channel was used as a model voltage-gated channel to probe the interaction of volatile general anesthetics with gating mechanisms. The effects of three anesthetics, chloroform (CHCl3), isoflurane, and halothane, were studied using recombinant native and mutant Shaker channels expressed in Xenopus oocytes. Gating currents and macroscopic ionic currents were recorded with the cut-open oocyte voltage-clamp technique. The effects of CHCl3 and isoflurane on gating kinetics of noninactivating mutants were opposite, whereas halothane had no effect. The effects on ionic currents were also agent dependent: CHCl3 and halothane produced a reduction of the macroscopic conductance, whereas isoflurane increased it. The results indicate that the gating machinery of the channel is mostly insensitive to the anesthetics during activation until near the open state. The effects on the conductance are mainly due to changes in the transitions in and out of the open state. The data give support to direct protein-anesthetic interactions. The magnitude and nature of the effects invite reconsideration of Shaker-like K+ channels as important sites of action of general anesthetics.

Studies on the Behavior Some Paints in Electro-insulating Fluid Based on Vegetable Esters

2018 ◽  
Vol 69 (5) ◽  
pp. 1139-1144
Author(s):  
Iosif Lingvay ◽  
Adriana Mariana Bors ◽  
Livia Carmen Ungureanu ◽  
Valerica Stanoi ◽  
Traian Rus
Keyword(s):  
Structural Changes ◽  
Oxidation Processes ◽  
High Oleic ◽  
Painting Materials ◽  
Insulating Paper ◽  
Different Types ◽  
Mechanism And Kinetics ◽  
Kinetics Of ◽  
Natural Ester

For the purpose of using three different types of painting materials for the inner protection of the transformer vats, their behavior was studied under actual conditions of operation in the transformer (thermal stress in electro-insulating fluid based on the natural ester in contact with copper for electro-technical use and electro-insulating paper). By comparing determination of the content in furans products (HPLC technique) and gases formed (by gas-chromatography) in the electro-insulating fluid (natural ester with high oleic content) thermally aged at 130 �C to 1000 hours in closed glass vessels, it have been found that the presence the investigated painting materials lead to a change in the mechanism and kinetics of the thermo-oxidation processes. These changes are supported by oxygen dissolved in oil, what leads to decrease both to gases formation CO2, CO, H2, CH4, C2H4 and C2H6) and furans products (5-HMF, 2-FOL, 2 -FAL and 2-ACF). The painting materials investigated during the heat treatment applied did not suffer any remarkable structural changes affecting their functionality in the electro-insulating fluid based on vegetable esters.

Biofilm Structure – An Important and Neglected Parameter in Waste Water Treatment

1989 ◽  
Vol 21 (8-9) ◽  
pp. 805-814 ◽  
Author(s):  
F. R. Christensen ◽  
G. Holm Kristensen ◽  
J. la Cour Jansen
Keyword(s):  
Wastewater Treatment ◽  
Structural Changes ◽  
Waste Water Treatment ◽  
Experimental Investigations ◽  
Biofilm Reactors ◽  
Substrate Removal ◽  
Treatment Processes ◽  
Laboratory Reactor ◽  
Biofilm Structure ◽  
Kinetics Of

Experimental investigations on the kinetics of wastewater treatment processes in biofilms were performed in a laboratory reactor. Parallel with the kinetic experiments, the influence of the biofilm kinetics on the biofilm structure was studied at macroscopic and microscopic levels. The close interrelationship between biofilm kinetics and structural changes caused by the kinetics is illustrated by several examples. From the study, it is evident that the traditional modelling of wastewater treatment processes in biofilm reactors based on substrate removal kinetics alone will fail in many cases, due to the inevitable changes in the biofilm structure not taken into consideration. Therefore design rules for substrate removal in biofilms used for wastewater treatment must include correlations between the removal kinetics and the structure and development of the biological film.

scholarly journals The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

2016 ◽  
Vol 27 (24) ◽  
pp. 3791-3799 ◽  
Author(s):  
Hafida Sellou ◽  
Théo Lebeaupin ◽  
Catherine Chapuis ◽  
Rebecca Smith ◽  
Anna Hegele ◽  
...  
Keyword(s):  
Dna Damage ◽  
Structural Changes ◽  
Cellular Responses ◽  
Dna Lesions ◽  
Fast Kinetics ◽  
Important Player ◽  
Chromatin Relaxation ◽  
Kinetics Of ◽  
Laser Microirradiation

Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo.

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