A Novel Fluorescence Assay for Voltage-Gated Ion Channels Based upon Light Induced Voltage Clamp

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Ion channels are a key target class with a high therapeutic potential in virtually all possible disease indications. In addition, a potential side effect of pharmaceutical compounds is the blocking of hERG channels in heart cells making easy and cost effective hERG safety screening necessary for drug development today. 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 stages of drug discovery.Here we demonstrate a method that allows light-induced activation of voltage-gated ion channels and the concurrent imaging of membrane potential changes using voltage-sensitive dyes. This light-induced voltage clamp (LIVC) method uses photostimulation through channelrhodopsin-2 (ChR2) to activate voltage-gated ion channels. ChR2 allows light to be immediately transduced into a depolarizing ionic current, which in turn causes voltage-gated ion channels to open. In our system we coexpressed ChR2 either with the voltage-gated potassium channels hERG or hKv1.5 in cell lines and in Xenopus oocytes. In electrophysiological experiments we show that light-induced depolarization through ChR2 sufficed to activate hERG as well as hKv1.5 channels. We were further able to optically monitor the light-induced membrane de- and hyperpolarizations on a millisecond timescale with the voltage-sensitive RH421 and Annine6. The fluorescence readout reflected the dose-response relationships of the hERG blocker Terfenadine and the hKv1.5 inhibitor DPO-1 obtained from patch-clamp measurements.LIVC represents a solely optical technology with remote activation of the target voltage-gated ion channels 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 high temporal resolution, membrane potential control and repetitive stimulation.