Resolving the open state enigma of TRPC3 by photopharmacology

Ion channels regulate cellular homeostasis by transporting various ions in and out of the cell. The majority of cellular processes are controlled particularly by flow of calcium into the cell causing fluctuations in the calcium concentration. Transient Receptor Potential Canonical 3 (TRPC3) channel is one of the calcium-permeable proteins in the plasma membrane and is highly present in human brain. In health, this channel regulates function of the brain regions responsible for a proper movement coordination. Recently hyperactivity of TRPC3 based on a pathological mutation has been found in a patient with cerebellar ataxia and linked to disease. This finding sets TRPC3 as a novel target in treatment of movement disorders in humans. Therefore, it is of high priority to develop pharmacological tools to control this channel that can lead to an improvement of the wellbeing of patients with movement neuropathologies. In order to develop precise modulators of a protein, it is necessary to determine its structure. Single particle cryogenic microscopy (cryo-EM) allows for a specimen of interest to be visualized at a very high resolution and precision. This technique enables to resolve protein structure and to predict chemical structures that are the likely to bind to the protein. Importantly, ion channels have different functional states: resting (without modulator), modulator-activated and modulator-inhibited. The protein structure of each of these functional states differs and depends on the pharmacological agent it is exposed to. Thus, cryo-EM of the TRPC3 has to be done in both absence and presence of the modulators. The main obstacle for application of pharmacological agents during cryo-EM analysis is the poor control over their concentration next to the protein of interest. Recently, it has become possible to control modulators that bind to TRPC3 channels effectively by specific wavelength. This method of light-mediated control of proteins is assigned photopharmacology. In our laboratory, we have developed and characterized photomodulators that enabled control of the TRPC3 functional states by light with a high temporal and spatial precision. We are confident that these photoagents will help us to resolve the functional states of TRPC3 during cryo-EM. Therefore, the main aim of this project is to resolve TRPC3 structure using cryo-EM and to determine the possible interaction sites for pharmacological agents using photopharmacology. This project will set the basis for a novel pharmacological intervention in the treatment of cerebellar ataxia.