Abstract
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.
Highlights
Most small-molecule sodium channel inhibitors bind to the local anesthetic binding site, and they are strongly state-dependent, showing ∼10-fold–1,000-fold higher affinity to inactivated channels (Lenkey et al, 2011)
This is the basis of the well-known difference between subclasses of class I antiarrhythmics, but binding/unbinding kinetics is important in the therapy of hyperexcitability-related skeletal muscle disorders (Simkin, 2011; Cannon, 2018), as well as diseases of the peripheral and central nervous system, such as certain pain syndromes and epilepsies
To understand the effect of drugs on ion channels, one must study the complex kinetics of ion channel gating, the complex dynamics of drug distribution, as well as the multiple interactions between the two
Summary
Most small-molecule sodium channel inhibitors bind to the local anesthetic binding site, and they are strongly state-dependent, showing ∼10-fold–1,000-fold higher affinity to inactivated channels (Lenkey et al, 2011) For this reason, as it has long been recognized, determining an IC50 value with a single voltage protocol means practically nothing. Sodium Channel Inhibitor Binding Dynamics different holding potentials, and determining the shift of steadystate availability curves at different drug concentrations are essentially equivalent experiments, as it has been discussed before—see Figure 1 of Lenkey et al (2011) It is a general practice, that instead of a single IC50 value, the resting-state-, and inactivated-state-affinities (KR and KI) are given for individual compounds. The last process may be further delayed if the compound has accumulated within intracellular lipid compartments, the depletion of which might require a longer time
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