Optogenetic and optical probing of TREK channel physiology – OPERAS

Maintenance of a negative resting membrane potential underlies the basis of neuronal excitability. This negative potential is generated by a potassium-selective leak (or background) current, which is mediated by K2P channels. K2P channels are sensitive to a wide variety of stimuli including lipids, mechanical stretch and intra- and extra-cellular acidification and are notably involved in mood regulation. The absence of specific pharmacology renders functional and structural studies of K2P channels difficult. To overcome this limitation, I recently developed a TREK1 channel that is controlled by light via a tethered photoisomerizable pore-blocker. This photoswitchable channel, called TREKlight, behaves like a wild-type channel in visible light but a pulse of UV light reversibly closes it. To address the role of TREK1 in hippocampal neurons in culture, I developed, based on TREKlight, a light-gated dominant-negative form of TREK1 (TREK1-PCS) which allows natively expressed channels to be targeted and reversibly regulated by light. OPERAS aims to further extend this technique to study TREK channel function and subunit organization. First, we will produce Knock-In mice in which the TREK1 channel is replaced by TREKlight (StarTREK mouse). StarTREK mice will have the same characteristics as wild type mice but with the additional advantage that its TREK channels can be instantaneously and reversibly blocked with light with high temporal and spatial precision, in order to address the function of the channel in several paradigms. Secondly, by combining TREK1-PCS and a single molecule pull-down assay (SiMPull), we will be able to determine the structural/pharmacological and functional impact of TREK1 heteromerization. Since these approaches offer a powerful general strategy to gain photocontrol over a large collection of channels/receptors, we will next apply them for obtaining a pharmacological foothold and to characterize subunit organization in other K2P channels.