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.

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 Electrophysiological and Pharmacological Characterization of Human Inwardly Rectifying Kir2.1 Channels on an Automated Patch-Clamp Platform

2019 ◽  
Vol 17 (3) ◽  
pp. 89-99 ◽  
Author(s):  
Camille Sanson ◽  
Brigitte Schombert ◽  
Bruno Filoche-Rommé ◽  
Michel Partiseti ◽  
G. Andrees Bohme
Keyword(s):  
Patch Clamp ◽  
Pharmacological Characterization ◽  
Automated Patch Clamp ◽  
Inwardly Rectifying

scholarly journals Characterization of NaV1.8 on a Highly Parallel Automated Patch Clamp System

2016 ◽  
Vol 110 (3) ◽  
pp. 113a
Author(s):  
Markus Rapedius ◽  
Andrea Bruggemann ◽  
Tom Goetze ◽  
Claudia Haarmann ◽  
Ilka Rinke ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Automated Patch Clamp

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.

scholarly journals Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chai-Ann Ng ◽  
Jessica Farr ◽  
Paul Young ◽  
Monique J Windley ◽  
Matthew D Perry ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Lines ◽  
High Throughput ◽  
Rapid Assessment ◽  
Cost Effective ◽  
Missense Variant ◽  
Loss Of Function ◽  
Channel Expression ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology

Abstract KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

scholarly journals An Advanced Automated Patch Clamp Protocol Design to Investigate Drug—Ion Channel Binding Dynamics

2021 ◽  
Vol 12 ◽  
Author(s):  
Peter Lukacs ◽  
Krisztina Pesti ◽  
Mátyás C. Földi ◽  
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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