An advanced automated patch clamp protocol design to investigate drug – ion channel binding dynamics

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.

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

“…In the accompanying paper (Lukacs et al, 2021) we describe how different degrees of inhibition observed in the 17-pulse protocol can be interpreted as revealing the state-dependent onset (SDO; how the inhibition depended on the length of depolarizations), the recovery from inactivation (RFI; how the inhibition depended on the length of hyperpolarizations), and steady-state inactivation (SSI; how the inhibition depended on the membrane potential). We have distinguished "macrodynamics," which was the onset/offset upon drug perfusion and removal, and occurred on the second-timescale; and "micro-dynamics," which was the onset/offset upon conformational transitions of the channel population, and occurred on the millisecond timescale.…”

“…In addition to automatic fitting, described in the accompanying paper (Lukacs et al, 2021), in this study we used a different approach to process SDO, RFI, and SSI data. This analysis focused on deriving compound-specific but concentration-independent descriptors of the mechanism of action, which can lead to a more thorough understanding of the sub-processes involved in drug action, and which can later serve as a basis for constructing kinetic models for the simulation of drug-specific effects.…”

“…Cell culture and electrophysiology were done as described in the accompanying paper (Lukacs et al, 2021). The recombinant rNaV1.4 channel-expressing cell line was generated as described before (Lukacs et al, 2018).…”

“…We used a complex 17-pulse voltage protocol (Figure 1A), described in detail in the accompanying paper (Lukacs et al, 2021), which allowed the assessment of gating kinetics and gating equilibrium in the absence and the presence of inhibitor/ modulator compounds. The protocol investigated the effect of different durations of depolarizations: 2.5, 7.5, 22.5, and 67.5 ms with 2.5 ms hyperpolarizing gaps between them; this section (pulses #1 to #5) is shown by different shades of green in Figure 1, and was named "state-dependent onset" (SDO).…”

“…We termed this process "microdynamics," to distinguish from the onset and offset of drug effects upon perfusion and washout of riluzole ("macrodynamics"), which occurred on a ∼1000-fold slower time scale (Figure 1B, left panel; Table 1). For a more detailed discussion of micro-and macro-dynamics see the accompanying paper (Lukacs et al, 2021).…”