scholarly journals The activation gate controls steady-state inactivation and recovery from inactivation in Shaker

2020 ◽  
Vol 152 (8) ◽  
Author(s):  
Tibor G. Szanto ◽  
Florina Zakany ◽  
Ferenc Papp ◽  
Zoltan Varga ◽  
Carol J. Deutsch ◽  
...  
Keyword(s):  
Steady State ◽  
Membrane Potentials ◽  
Tight Coupling ◽  
Shaker Channels ◽  
Recovery From Inactivation ◽  
Activation Gate ◽  
The Status ◽  
Steady State Inactivation

Despite major advances in the structure determination of ion channels, the sequence of molecular rearrangements at negative membrane potentials in voltage-gated potassium channels of the Shaker family remains unknown. Four major composite gating states are documented during the gating process: closed (C), open (O), open-inactivated (OI), and closed-inactivated (CI). Although many steps in the gating cycle have been clarified experimentally, the development of steady-state inactivation at negative membrane potentials and mandatory gating transitions for recovery from inactivation have not been elucidated. In this study, we exploit the biophysical properties of Shaker-IR mutants T449A/V474C and T449A/V476C to evaluate the status of the activation and inactivation gates during steady-state inactivation and upon locking the channel open with intracellular Cd2+. We conclude that at negative membrane potentials, the gating scheme of Shaker channels can be refined in two aspects. First, the most likely pathway for the development of steady-state inactivation is C→O→OI⇌CI. Second, the OI→CI transition is a prerequisite for recovery from inactivation. These findings are in accordance with the widely accepted view that tight coupling is present between the activation and C-type inactivation gates in Shaker and underscore the role of steady-state inactivation and recovery from inactivation as determinants of excitability.

Role of S4 positively charged residues in the regulation of Kv4.3 inactivation and recovery

2007 ◽  
Vol 293 (3) ◽  
pp. C906-C914 ◽  
Author(s):  
Matthew R. Skerritt ◽  
Donald L. Campbell
Keyword(s):  
Positive Charge ◽  
Voltage Sensitivity ◽  
Shaker Channels ◽  
Recovery From Inactivation ◽  
Kv4 Channels ◽  
Charged Residues ◽  
Arginine Residues ◽  
Voltage Sensing Domain ◽  
Positively Charged

The molecular and biophysical mechanisms by which voltage-sensitive K+ (Kv)4 channels inactivate and recover from inactivation are presently unresolved. There is a general consensus, however, that Shaker-like N- and P/C-type mechanisms are likely not involved. Kv4 channels also display prominent inactivation from preactivated closed states [closed-state inactivation (CSI)], a process that appears to be absent in Shaker channels. As in Shaker channels, voltage sensitivity in Kv4 channels is thought to be conferred by positively charged residues localized to the fourth transmembrane segment (S4) of the voltage-sensing domain. To investigate the role of S4 positive charge in Kv4.3 gating transitions, we analyzed the effects of charge elimination at each positively charged arginine (R) residue by mutation to the uncharged residue alanine (A). We first demonstrated that R290A, R293A, R296A, and R302A mutants each alter basic activation characteristics consistent with positive charge removal. We then found strong evidence that recovery from inactivation is coupled to deactivation, showed that the precise location of the arginine residues within S4 plays an important role in the degree of development of CSI and recovery from CSI, and demonstrated that the development of CSI can be sequentially uncoupled from activation by R296A, specifically. Taken together, these results extend our current understanding of Kv4.3 gating transitions.

Abstract 20627: Modulation of Cardiac Sodium Channel by Palmitoylation and Its Association With Cardiac Disease Mutations

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Zifan Pei ◽  
Andy Hudmon ◽  
Theodore R Cummins
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Functional Activity ◽  
Site Directed Mutagenesis ◽  
Significant Shift ◽  
Biophysical Properties ◽  
Disease Mutations ◽  
Cardiac Sodium Channel ◽  
Steady State Inactivation

Cardiac sodium channel (Nav1.5) is responsible for the generation and propagation of the cardiac action potential, which underlies cardiac excitability. It can be modified by a variety of post-translational modifications. Palmitoylation is one of the most common post-translational lipid modifications that can dynamically regulate protein life cycle and functional activity. In our study, we identified palmitoylation on Nav1.5 and its alteration in channel biophysical properties. Nav1.5 palmitoylation was identified in both HEK 293 cells stably expressing Nav1.5 and cardiac tissues using acyl-biotin exchange assay. Nav1.5 palmitoylation was inhibited by pre-incubating the cells with the inhibitor 2-Br-Palmitate (2BP, 25uM, 24hrs). Biophysically, 2BP treatment drastically shifted the channel steady-state inactivation to more hyperpolarized voltages, suggesting palmitoylation altering channel functional activity. In addition, four predicted endogenous palmitoylation sites were identified using CSS-Palm 3.0. Site-directed mutagenesis method was used to generate a cysteine removing background of wt Nav1.5 to study the role of predicted sites. Patch clamp analysis of wt and cysteine-removed Nav1.5 revealed a significant change in channel biophysics. 2BP treatment significantly shifted steady-state inactivation of wt Nav1.5 while not affecting cysteine-removed Nav1.5 significantly, indicating the important role of these four cysteine sites in modulating channel palmitoylation. Moreover, several LQT disease mutations were identified to potentially add or remove palmitoylation sites. Further analysis of these disease mutations revealed a significant shift in channel steady-state inactivation and this alteration cannot be seen with the substitution of other residues on the same site, suggesting the specific role of cysteine residue in causing the functional alteration. For the LQT mutation that removes potential palmitoylation site, 2BP treatment did not affect channel biophysical properties, indicating the essential role of this cysteine in channel palmitoylation. These results suggest that palmitoylation on Nav1.5 regulates channel functional activity and its modulation may contribute to new cardiac channelopathies.

The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6 voltage-dependent sodium channel

2007 ◽  
Vol 293 (2) ◽  
pp. C783-C789 ◽  
Author(s):  
Christian Rosker ◽  
Birgit Lohberger ◽  
Doris Hofer ◽  
Bibiane Steinecker ◽  
Stefan Quasthoff ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channels ◽  
Therapeutic Intervention ◽  
Time Course ◽  
Specific Interaction ◽  
Xenopus Laevis Oocytes ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Invaluable Tool

The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Nav1.8). Generally, TTX was 40–231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Nav1.6, where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC50 values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 ± 1.3/1,260 ± 121 (Nav1.2), 2.8 ± 2.3/341 ± 36 (Nav1.3), 4.5 ± 1.0/988 ± 62 (Nav1.4), 1,970 ± 565/78,500 ± 11,600 (Nav1.5), 3.8 ± 1.5/7.8 ± 2.3 (Nav1.6), 5.5 ± 1.4/1,270 ± 251 (Nav1.7), and 1,330 ± 459/>30,000 (Nav1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Nav1.6 subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Nav1.5 subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Nav1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Nav1.6-mediated function, but also for therapeutic intervention.

Electrophysiological characteristics of cloned skeletal and cardiac muscle sodium channels

1996 ◽  
Vol 271 (2) ◽  
pp. H498-H506 ◽  
Author(s):  
M. Chahine ◽  
I. Deschene ◽  
L. Q. Chen ◽  
R. G. Kallen
Keyword(s):  
Steady State ◽  
Sodium Channels ◽  
H Infinity ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Current Decay ◽  
Voltage Gated Sodium Channels ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Kinetics Of

The alpha-subunit encoding for voltage-gated sodium channels rSkM1 (rat skeletal muscle subtype 1) and hH1 (human heart subtype 1) has been cloned and expressed by various groups under various conditions in Xenopus oocytes and the tsA201 (HEK 293) mammalian cell line derived from human embryonic kidney cells. In this study, we have expressed hH1 and rSkM1 in tsA201 cells for comparison under the same conditions using patch-clamp methods. Our results show significant differences in the current-voltage (I-V) relationship, kinetics of current decay, voltage dependence of steady-state inactivation, and the time constant for recovery from inactivation. We studied several rSkM1/hH1 chimeric sodium channels to identify the structural regions responsible for the different biophysical behavior of the two channel subtypes. Exchanging the interdomain (ID3-4) loops, thought to contain the inactivation particle, between rSkM1 and hH1 had no effect on the electrophysiological behaviors, including inactivation, indicating that the differences in channel subtype characteristics are determined by parts of the channel other than the ID3-4 segment. The data on a chimeric channel in which D1 and D4 are derived from hH1 while D2 and D3 and the ID1-2, ID2-3, and ID3-4 loops are from rSkM1 show that D1 and/or D4 seem to be responsible for the slower kinetics of inactivation of hH1 while D2 and/or D3 appear to contain the determinants for the differences in the I-V relationship, steady-state inactivation (h infinity) curve, and the kinetics of the recovery from inactivation.

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4

2006 ◽  
Vol 291 (5) ◽  
pp. C966-C976 ◽  
Author(s):  
Hong-Ling Li ◽  
Yu-Jie Qu ◽  
Yi Chun Lu ◽  
Vladimir E. Bondarenko ◽  
Shimin Wang ◽  
...  
Keyword(s):  
Steady State ◽  
Interacting Protein ◽  
Voltage Gated ◽  
Time To Peak ◽  
Kv Channel ◽  
Closed State ◽  
Channel Inactivation ◽  
Recovery From Inactivation

Voltage-gated K+ channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K+ currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At −60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K+ channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K+ channels.

scholarly journals p.L1612P, a Novel Voltage-gated Sodium Channel Nav1.7 Mutation Inducing a Cold Sensitive Paroxysmal Extreme Pain Disorder

2015 ◽  
Vol 122 (2) ◽  
pp. 414-423 ◽  
Author(s):  
Marc R. Suter ◽  
Zahurul A. Bhuiyan ◽  
Cédric J. Laedermann ◽  
Thierry Kuntzer ◽  
Muriel Schaller ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Cold Exposure ◽  
Pain Disorder ◽  
Clinical Genetic ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Extreme Pain

Abstract Background: Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. Methods: Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. Results: The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady–state inactivation curve (V1/2 from −61.8 ± 4.5 mV to −30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady–state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. Conclusions: The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.

scholarly journals DISORIENTASI PENDIDIKAN MADRASAH DI INDONESIA

2017 ◽  
Vol 3 (1) ◽  
pp. 61
Author(s):  
Hasan Basri
Keyword(s):  
School Culture ◽  
Secular Education ◽  
The Family ◽  
The Status ◽  
The Will ◽  
And Function ◽  
Changing Schools ◽  
Further Development

Madrasah in the Middle East has known eight or nine centuries before madrasah in Indonesia, which emerged as a reaction to the reform movement as well as a response to the policy of Dutch colonizers secular education. Madrasah got a decent place in Indonesia after rising SKB 3 minister (Minister of Interior, Minister of Education and Culture, and the Minister of Religious Affairs) in 1975, where madrasas equated with other schools in terms of the status of the diploma, graduates continuing education opportunities and changing schools. In a further development, the school as disoriented. It is caused by two things: first, a paradigm shift towards sekularistik. Education implementation has marred even be interpreted as a partial instead of a holistic paradigm as desired by Islam. Supposedly, the madrasa education as a whole should make Islam as a principle in the determination of educational objectives, the formulation of the curriculum and standard of value of science and the learning process, including determining the qualifications of teachers and school culture that will be developed in the madrasas. Second, the functional institutional weakness as a result of shifting the orientation and function of the family and their influence and societal demands materialistic-hedonistic.The weakness seen in a mess madrasa curriculum, not optimal role of teachers as well as school culture that is not in line with the will of Islam.

scholarly journals Analisa Kerjasama Pihak Corporate dengan Masyarakat dalam Pengembangan Pariwisata di Desa Singapadu Kaler, Kabupaten Gianyar

2020 ◽  
Vol 8 (1) ◽  
pp. 125
Author(s):  
Claudia Grace Kusumawardani ◽  
Putri Kusuma Sanjiwani
Keyword(s):  
Qualitative Method ◽  
Secondary Data ◽  
Sampling Procedure ◽  
Analysis Techniques ◽  
Community Tourism ◽  
The Status ◽  
Corporate Community ◽  
Depth Interviews

In village tourism developing, it is necessary to have cooperation carried out by tourism Stakeholders, both Government, Community and Entrepreneurs or private parties. The collaboration must be balanced according to the status and role of each stakeholder so that harmonious cooperation can be created that is impartial to anyone. The research method used is a qualitative method with qualitative data analysis techniques, The source of data from this study comes from primary and secondary data. Data collection techniques are carried out by observation, in-depth interviews and documentation. Determination of informants is done by purposive sampling procedure. The results of this study indicate that based on the characteristics, tasks, objectives and functions of the BUMDES ( Badan Usaha Milik Desa ) are still not optimal, it can be seen that a number of things that have not yet fulfilled and still need to be reviewed so that BUMDES ( Badan Usaha Milik Desa) can collaborate and coordinate tourism village units optimally. Keywords: Corporate, Community, Tourism Development

The Role of Sodium in the Conversion of Pyruvate to Phosphoenolpyruvate in Mesophyll Chloroplasts of C4 Plants

1996 ◽  
Vol 23 (2) ◽  
pp. 171 ◽  
Author(s):  
PF Brownell ◽  
LM Bielig
Keyword(s):  
Oxygen Evolution ◽  
Malic Enzyme ◽  
Small Samples ◽  
Panicum Miliaceum ◽  
Tight Coupling ◽  
Irreversible Damage ◽  
C4 Species ◽  
The Hill

PEP formation from pyruvate was determined in mesophyll chloroplasts mechanically isolated from sodium-deficient and sodium-replete plants of the NADP malic enzyme-type C4 species, Kochia trichophylla. An extremely sensitive method for estimating PEP was developed which permitted determination of picomole quantities of PEP in small samples taken sequentially from the mesophyll chloroplast suspension concurrently with observations on oxygen evolution. It was shown that PEP formation requires light and depends upon the intactness of the chloroplasts. The rate of formation of PEP from pyruvate increased in the presence of the Hill reagent, oxaloacetate, thus indicating its dependence on non-cyclic photophosphorylation for the supply of ATP required in the conversion of pyruvate to PEP. The optimum inorganic phosphate concentration for PEP formation was approximately 16 mM. The rates of oxygen evolution and PEP formation were equivalent at concentrations of pyruvate up to 20 mM, suggesting tight coupling between electron transport and phosphorylation. In both Kochia trichophylla and the NAD malic enzyme-type, Panicum miliaceum, the rates of PEP formation were greater in the mesophyll chloroplasts from sodium-replete than from sodium-deficient plants. Chloroplasts resuspended in 'sodium-free'media containing less than 30 μM (0.7 ppm) sodium showed reduced rates of PEP formation compared with chloroplasts resuspended in media to which 1.0 mM (23 ppm) sodium had been added. Both media contained 10 mM 'sodium-free' KCI. These results indicate that sodium ions may be required to maintain the functional integrity of the mesophyll chloroplasts and that irreversible damage is sustained when sodium is absent during their isolation.

Sign in / Sign up

Export Citation Format

Share Document