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.

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 Publisher Correction: Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
James Kramer ◽  
Herbert M. Himmel ◽  
Anders Lindqvist ◽  
Sonja Stoelzle-Feix ◽  
Khuram W. Chaudhary ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Drug Effects ◽  
Recombinant Cells ◽  
Cross Platform ◽  
Automated Patch Clamp ◽  
Cross Site

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 Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
James Kramer ◽  
Herbert M. Himmel ◽  
Anders Lindqvist ◽  
Sonja Stoelzle-Feix ◽  
Khuram W. Chaudhary ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Drug Effects ◽  
Recombinant Cells ◽  
Cross Platform ◽  
Automated Patch Clamp ◽  
Cross Site

High-Throughput Screening of NaV1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument

2016 ◽  
Vol 14 (2) ◽  
pp. 93-108 ◽  
Author(s):  
Chris Chambers ◽  
Ian Witton ◽  
Cathryn Adams ◽  
Luke Marrington ◽  
Juha Kammonen
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
High Throughput Screening ◽  
Automated Patch Clamp

scholarly journals Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module

2015 ◽  
Vol 21 (6) ◽  
pp. 779-793 ◽  
Author(s):  
A. Obergrussberger ◽  
A. Bru ggemann ◽  
T. A. Goetze ◽  
M. Rapedius ◽  
C. Haarmann ◽  
...  
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
High Throughput Screening ◽  
Automated Patch Clamp

scholarly journals MATLAB-based automated patch-clamp system for awake behaving mice

2015 ◽  
Vol 114 (2) ◽  
pp. 1331-1345 ◽  
Author(s):  
Niraj S. Desai ◽  
Jennifer J. Siegel ◽  
William Taylor ◽  
Raymond A. Chitwood ◽  
Daniel Johnston
Keyword(s):  
Patch Clamp ◽  
High Throughput Screening ◽  
Automated System ◽  
Electrode Impedance ◽  
Seamless Integration ◽  
Use Of Time ◽  
Good Target ◽  
Instrument Control ◽  
Automated Patch Clamp

Automation has been an important part of biomedical research for decades, and the use of automated and robotic systems is now standard for such tasks as DNA sequencing, microfluidics, and high-throughput screening. Recently, Kodandaramaiah and colleagues ( Nat Methods 9: 585–587, 2012) demonstrated, using anesthetized animals, the feasibility of automating blind patch-clamp recordings in vivo. Blind patch is a good target for automation because it is a complex yet highly stereotyped process that revolves around analysis of a single signal (electrode impedance) and movement along a single axis. Here, we introduce an automated system for blind patch-clamp recordings from awake, head-fixed mice running on a wheel. In its design, we were guided by 3 requirements: easy-to-use and easy-to-modify software; seamless integration of behavioral equipment; and efficient use of time. The resulting system employs equipment that is standard for patch recording rigs, moderately priced, or simple to make. It is written entirely in MATLAB, a programming environment that has an enormous user base in the neuroscience community and many available resources for analysis and instrument control. Using this system, we obtained 19 whole cell patch recordings from neurons in the prefrontal cortex of awake mice, aged 8–9 wk. Successful recordings had series resistances that averaged 52 ± 4 MΩ and required 5.7 ± 0.6 attempts to obtain. These numbers are comparable with those of experienced electrophysiologists working manually, and this system, written in a simple and familiar language, will be useful to many cellular electrophysiologists who wish to study awake behaving mice.

Sign in / Sign up

Export Citation Format

Share Document