scholarly journals Compound-specific, concentration-independent biophysical properties of sodium channel inhibitor mechanism of action

2021 ◽  
Author(s):  
Krisztina Pesti ◽  
Matyas C Foldi ◽  
Katalin Zboray ◽  
Adam V Toth ◽  
Peter Lukacs ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Mechanism Of Action ◽  
Kinetic Properties ◽  
Analysis Method ◽  
Experimental Protocol ◽  
Biophysical Properties ◽  
Recovery From Inactivation ◽  
Automated Patch Clamp ◽  
Inhibitor Compounds ◽  
Onset Rate

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

scholarly journals Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology

2021 ◽  
Vol 12 ◽  
Author(s):  
Krisztina Pesti ◽  
Mátyás C. Földi ◽  
Katalin Zboray ◽  
Adam V. Toth ◽  
Peter Lukacs ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Sodium Channel ◽  
Mechanism Of Action ◽  
Kinetic Properties ◽  
Biophysical Properties ◽  
Recovery From Inactivation ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology ◽  
Inhibitor Compounds

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

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.

scholarly journals Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes

2019 ◽  
Vol 20 (20) ◽  
pp. 5083 ◽  
Author(s):  
Sonia Arilla-Luna ◽  
Ana Serrano ◽  
Milagros Medina
Keyword(s):  
Binding Site ◽  
Thermodynamic Parameters ◽  
Spectroscopic Properties ◽  
Open Cavity ◽  
Flavin Mononucleotide ◽  
Kinetic Properties ◽  
Biophysical Properties ◽  
Conserved Motif ◽  
Corynebacterium Ammoniagenes ◽  
Optimal Geometry

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.

scholarly journals The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides

2016 ◽  
Vol 291 (38) ◽  
pp. 20113-20124 ◽  
Author(s):  
Yongqiang Zhang ◽  
Yuzhe Du ◽  
Dingxin Jiang ◽  
Caitlyn Behnke ◽  
Yoshiko Nomura ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Mechanism Of Action ◽  
Channel Blocker ◽  
Receptor Site ◽  
Sodium Channel Blocker

scholarly journals Sodium channel activators: Model of binding inside the pore and a possible mechanism of action

FEBS Letters ◽  
2005 ◽  
Vol 579 (20) ◽  
pp. 4207-4212 ◽  
Author(s):  
Denis B. Tikhonov ◽  
Boris S. Zhorov
Keyword(s):  
Sodium Channel ◽  
Mechanism Of Action

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 Two functionally distinct 4-aminopyridine-sensitive outward K+ currents in rat atrial myocytes.

1992 ◽  
Vol 100 (6) ◽  
pp. 1041-1067 ◽  
Author(s):  
W A Boyle ◽  
J M Nerbonne
Keyword(s):  
Experimental Testing ◽  
Potential Range ◽  
Kinetic Properties ◽  
Atrial Myocytes ◽  
Adult Rat ◽  
Outward Currents ◽  
Recovery From Inactivation ◽  
Functional Inactivation ◽  
The Individual ◽  
K Currents

In the experiments here, the detailed kinetic properties of the Ca(2+)-independent, depolarization-activated outward currents (Iout) in enzymatically dispersed adult rat atrial myocytes were studied. Although there is only slight attenuation of peak Iout during brief (100 ms) voltage steps, substantial decay is evident during long (10 s) depolarizations. The analyses here reveal that current inactivation is best described by the sum of two exponential components, which we have termed IKf and IKs to denote the fast and slow components, respectively, of Iout decay. At all test potentials, IKf inactivates approximately 20-fold more rapidly than IKs. Neither the decay time constants nor the fraction of Iout remaining at the end of 10-s depolarizations varies over the potential range of 0 to +50 mV, indicating that the rates of inactivation and recovery from inactivation are voltage independent. IKf recovers from inactivation completely, independent of the recovery of IKs, and IKf recovers approximately 20 times faster than IKs. The pharmacological properties of IKf and IKs are similar: both components are sensitive to 4-aminopyridine (1-5 mM) and both are relatively resistant to externally applied tetraethylammonium (50 mM). Taken together, these findings suggest that IKf and IKs correspond to two functionally distinct K+ currents with similar voltage-dependent properties and pharmacologic sensitivities, but with markedly different rates of inactivation and recovery from inactivation. From the experimental data, several gating models were developed in which voltage-independent inactivation is coupled either to channel opening or to the activation of the individual channel subunits. Experimental testing of predictions of these models suggests that voltage-independent inactivation is coupled to activation, and that inactivation of only a single subunit is required to result in functional inactivation of the channels. This model closely approximates the properties of IKf and IKs, as well as the composite outward currents, measured in adult rat atrial myocytes.

scholarly journals An advanced automated patch clamp protocol design to investigate drug - ion channel binding dynamics.

2021 ◽  
Author(s):  
Peter Lukacs ◽  
Krisztina Pesti ◽  
Matyas C Foldi ◽  
Katalin Zboray ◽  
Adam V Toth ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Mechanism Of Action ◽  
Time Scale ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Neuromuscular Disorders ◽  
Rapid Assessment ◽  
Drug Effects ◽  
Dissociation Kinetics ◽  
Automated Patch Clamp

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases.

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