Parallel All-Optical Assay to Study Use-Dependent Functioning of Voltage-Gated Ion Channels in a Miniaturized Format

SLAS Discov. 2021 Mar;26(3):460-469. doi: 10.1177/2472555220976083. Epub 2020 Dec 17.

Abstract

Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.

Keywords: cardiotoxicity; high-throughput screening; ion channel; optogenetics; use dependency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algal Proteins / antagonists & inhibitors
  • Algal Proteins / genetics
  • Algal Proteins / metabolism
  • Biological Assay*
  • Calcium Channel Blockers / pharmacology*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism
  • Calcium Channels, L-Type / genetics
  • Calcium Channels, L-Type / metabolism
  • Cell Line
  • Chlamydomonas reinhardtii
  • Fluorescent Dyes / chemistry
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Ion Channel Gating / drug effects
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • NAV1.5 Voltage-Gated Sodium Channel / genetics
  • NAV1.5 Voltage-Gated Sodium Channel / metabolism
  • Nerve Tissue Proteins / antagonists & inhibitors
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Optical Imaging / methods
  • Optogenetics / methods*
  • Potassium Channel Blockers / pharmacology*
  • Potassium Channels, Inwardly Rectifying / antagonists & inhibitors
  • Potassium Channels, Inwardly Rectifying / genetics
  • Potassium Channels, Inwardly Rectifying / metabolism
  • Potassium Channels, Tandem Pore Domain / antagonists & inhibitors
  • Potassium Channels, Tandem Pore Domain / genetics
  • Potassium Channels, Tandem Pore Domain / metabolism
  • Rhodopsin / antagonists & inhibitors
  • Rhodopsin / genetics
  • Rhodopsin / metabolism

Substances

  • Algal Proteins
  • CACNA1D protein, human
  • CACNA2D1 protein, human
  • CACNB3 protein, human
  • Calcium Channel Blockers
  • Calcium Channels
  • Calcium Channels, L-Type
  • Fluorescent Dyes
  • Intracellular Signaling Peptides and Proteins
  • KCNJ4 protein, human
  • NAV1.5 Voltage-Gated Sodium Channel
  • Nerve Tissue Proteins
  • Potassium Channel Blockers
  • Potassium Channels, Inwardly Rectifying
  • Potassium Channels, Tandem Pore Domain
  • SCN5A protein, human
  • chlamyopsin protein, Chlamydomonas reinhardtii
  • potassium channel subfamily K member 3
  • Rhodopsin