A Novel Screening Tool for Voltage-Gated Ion Channels: Light Induced Voltage Clamp

Abstract

Ion channels are a key target class with a high therapeutic potential. Conventional screening techniques yield insufficient data quality particularly when assessing voltage-gated ion channels. Thus, the development of new reliable technologies is desirable to integrate ion channel screening into early lead generation of drug discovery.Here we demonstrate a method that allows non-invasive, millisecond light-induced activation of voltage-gated ion channels and the concurrent imaging of membrane potential changes using fast voltage-sensitive dyes. This light-induced voltage clamp method (LIVC) uses photostimulation through channelrhodopsin-2 (ChR2), to activate voltage-gated ion channels. ChR2 allows blue light (∼ 470 nm) to be immediately transduced into a depolarizing ionic current, which causes voltage-gated ion channels to open. We coexpressed ChR2 with the voltage-gated potassium channel hKv1.5 in HEK293 cells and in Xenopus oocytes. In electrophysiological experiments we show that the light-induced cell depolarization through ChR2 sufficed to open hKv1.5 channels; the light-induced membrane depolarization was greatly reduced with active hKv1.5 channels compared to the full ChR2 response during hKv1.5 inhibition. We were further able to optically monitor the light-induced membrane depolarizations on a millisecond timescale with the voltage-sensitive RH421. The fluorescence readout reflected the concentration-inhibition relationship of the hKv1.5 inhibitor DPO-1.LIVC represents a solely optical technology with remote activation of the target voltage-gated ion channels simply by the delivery of a flash of blue light and simultaneous detection of their activity employing voltage-sensitive dyes. It combines the high-throughput of optical methods with the high-content of patch clamp concerning and possible repetitive stimulation. Proof of concept and results from assay development for voltage-gated sodium and calcium channels as well as for the hERG channel underline the potential for LIVC to evolve into a high-throughput, high reliability assay for voltage-gated ion channels in general.