DUAL-COLOR APPROACHES FOR OPTOGENETIC HEARING RESTORATION

Optogenetic stimulation of spiral ganglion neurons is constrained by the kinetic and efficiency limitations of available channelrhodopsins, restricting the temporal fidelity at high frequency auditory encoding. Additionally, optical stimulation exhibits a reduced dynamic range compared to natural hearing. To address these challenges, we aim to develop dual-color optogenetic strategies combining protein engineering, spinning-disk confocal microscopy, and electrophysiological characterization. To improve temporal fidelity, we expressed two spectrally separated channelrhodopsins in a single cell. By sequentially activating f-Chrimson – a red-shifted depolarizing channelrhodopsin – and HcKCR2-f1, a blue-light-activated potassium-selective (hyperpolarizing) channelrhodopsin, we achieve accelerated photocurrent decay. This approach exploits the inverse relationship between current decay kinetics and charge transfer, enabling a flexible adjustment of photocurrent decay to maintain a level of charge transfer sufficient for reliable neuronal stimulation. To expand the dynamic range of optical stimulation, we seek to develop highly efficient, blue-light-activated actuators. We engineered novel non-potassium-selective (depolarizing) HcKCR2 variants that exhibit depolarizing responses, accelerated closing kinetics, and current densities matching state-of-the-art blue tools like Chronos and CatCh. Blue-light-activated channelrhodopsins optimized for efficiency expand the toolkit for dual-color optogenetic strategies, especially given their compatibility with red-shifted tools. When paired with red-light-activated actuators, the independent stimulation of different populations of spiral ganglion neurons may increase the dynamic range of optical stimulation. Together, these approaches represent a step toward next-generation optical cochlear implants capable of delivering more naturalistic auditory perception.