PT  - JOURNAL ARTICLE
AU  - Peter Lukacs
AU  - Krisztina Pesti
AU  - Mátyás C. Földi
AU  - Katalin Zboray
AU  - Adam V. Toth
AU  - Gábor Papp
AU  - Arpad Mike
TI  - An advanced automated patch clamp protocol design to investigate drug – ion channel binding dynamics
AID  - 10.1101/2021.07.05.451189
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.07.05.451189
4099  - http://biorxiv.org/content/early/2021/07/06/2021.07.05.451189.short
4100  - http://biorxiv.org/content/early/2021/07/06/2021.07.05.451189.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.SDO“state-dependent onset” protocolRFI“recovery from inactivation” protocolSSI“steady-state inactivation” protocol%RMSEpercentage root mean square errorErelrelative errorEIPeffective inhibitor potency