Ion channel trafficking studies using automated patch clamp technology

Abstract

Throughout the drug development process, one of the most common adverse effects leading to drug failure is cardiac arrhythmia. This condition arises from dysfunctions in the activity or expression of various ion channels that shape the cardiac action potential. Moreover, ion channel trafficking is a contributing factor in both inherited and acquired arrhythmias. Unlike the rapid effects of acute ion channel inhibition, disruptions in trafficking typically emerge over longer periods, ranging from several hours to days.

Several pharmacological agents, such as arsenic trioxide and pentamidine, can induce hERG channel deficiency by interfering with its biogenesis and intracellular trafficking.

A high-throughput assay for detecting drug-induced inhibition of hERG channel trafficking has been established using the SyncroPatch platform. This assay enables the identification of trafficking defects in recombinant cell lines following overnight drug exposure. The development and validation of the assay involved testing known therapeutic compounds (such as arsenic trioxide, amitriptyline, fluoxetine, pentamidine, amiodarone), and these drugs have been confirmed in this assay to cause hERG channel trafficking defects.

Briefly, 48 h prior to the experiment, cells resuspended in culture medium were plated in 384-well plates. After 24 h, compounds to be assessed as well as vehicle were added in the 384-well plates. Cells were incubated with compounds for 24 h. The day of the experiment, cells were detached from each well in the incubated assay plate and resuspended in the experimental solution. Evaluation of pre-treatment effects on the hERG current was possible thanks to the SyncroPatch, an automated patch clamp instrument appropriate for studying ion channels. Impairment of hERG channel function due to prolonged exposure of cells to drugs was evaluated measuring the alteration in peak hERG current amplitude following drug treatment compared to vehicle.

Understanding the mechanisms of drug-induced cardiotoxicity and differences in cardiac safety among drugs is key to developing safer compounds and better preclinical safety tests. The described assay, already adapted in our laboratories to the hNav1.5 sodium channel, highlights its broader applicability, as trafficking defects have been observed in multiple ion channels beyond hERG.