Highly Parallel All-Optical Real-Time Interrogation of Fast Voltage-Gated Ion Channels using Molecular Wire Voltage-Sensing Compounds

Abstract

Combining optogenetic control of cell membrane potential with real-time fluorescence-based voltage readouts has the potential to enable economical all-optical alternatives to patch clamp systems in screening large compound libraries against ion channel targets. Rapidly-inactivating voltage-gated ion channel targets are particularly challenging for high throughput optical screening because of the need for sub-millisecond scale temporal resolution, flexible voltage control, and highly parallel measurements. Here, we combine the instantaneous response of “molecular wire” voltage sensors with repetitive optical ion channel stimulation and simultaneous voltage readouts of multiple wells at 10 kHz sampling rate. Using cells engineered to express a channelrhodopsin optical actuator plus either the Nav1.5 or Nav1.7 sodium channel, induced action potentials were monitored at high S/N under regimens involving varying stimulation frequencies (0.5 – 8 Hz) or durations of inactivating membrane voltage potentials. Appropriate isoform selectivity as well as state and use-dependent pharmacology are shown for local anesthetic and voltage sensor-binding test compounds. Of note are results obtained with a “red-shifted” molecular wire probe at 660 nm excitation wavelength, allowing for completely independent voltage sensor monitoring and channelrhodopsin stimulation. The combination of optical sensors and actuators offer a promising platform for truly high throughput interrogation of ion channel targets that are difficult to screen using preexisting optical instrumentation.