Development and Engineering of a cGMP-gated K⁺ channel for Optogenetic Silencing

Abstract

Optogenetics enables researchers to control the activity of specific cells with remarkable precision using light. While many optogenetic tools are excellent at activating neurons, there are far fewer reliable tools for inhibiting neuronal activity, which is just as crucial for understanding brain function. One such tool, called PAC-K, is a two-component optogenetic inhibitory system composed of a photoactivated adenylyl cyclase and the bacterial potassium channel SthK. Although PAC-K performed very well in various tissues, some undesired side effects have been observed that limit its usefulness in the living animal. <br/> In this thesis, I aim to develop a better alternative: a tool that can silence excitatory cells with high specificity, minimal side effects, and reliable performance. I focused on modifying the SthK channel to make it respond selectively to cyclic GMP (cGMP). Through targeted mutagenesis, I engineered various candidate versions of the channel, referred to as SthK 1.0, 1.1, 2.0, and 2.1, which all respond to cGMP while being less responsive to cyclic AMP (cAMP). This improved cGMP sensitivity allows precise control of the channel using light-activated guanylyl cyclases, enabling effective and selective neuronal silencing. <br/> I characterized and validated the engineered channels using patch-clamp electrophysiology in HEK cells, confirming robust activation by cGMP with minimal response to cAMP. Next, I tested the system in vivo by expressing the CatRhGC together with SthK2.1 in the mouse hippocampus to investigate potential side effects. Using immunohistochemistry, I assessed markers such as GFAP (for astrocyte activation) and NeuN (for neuronal health), and found that our combined tool, now named RoCK 2.1, caused no noticeable toxicity or immune response compared to controls. <br/> In summary, this work introduces RoCK 2.1 as a new, safe, and highly effective optogenetic silencing tool. With its improved molecular precision, minimal side effects, and reliable performance, RoCK 2.1 holds strong potential to become a potent and widely applicable inhibitory optogenetic tool — particularly valuable in experiments where precise neuronal silencing is required without compromising cellular health or long-term tissue integrity.