Final Report Summary - TRPM3 IN SKIN (Cellular regulation of transient receptor potential melastatin 3 (TRPM3) and its role in skin sensation) The skin is not only the barrier of our body, but it is also a very important sensory organ collecting vital information from the external word. It has a central role in sensation of mechanical, chemical and thermal stimuli as well as pain or itch. These sensory functions are realized by the dense innervation of the skin. . On these sensory nerve endings, various molecules serve for the sensation and transmission of various external stimuli. Among this molecules, ion channels play an especially important role regulating excitability and action potential firing of the neurons. Ion channels of the transient receptor potential family (TRP channels) expressed by both sensory neurons and epithelial cells are generally considered as cellular sensors, due to their sensitivity to various physical stimuli and to diverse chemical ligands. These multimodal TRP channels (e.g. TRPV1, TRPA1 or TRPM8) have a central role in sensation of temperature, pain or itch and they are also appealing drug targets especially in pain associated syndromes. Recently, our group, the Laboratory of Ion Channel Research at the KU Leuven, reported that TRPM3, a less-known member of the melastatin subfamily of TRP channels, also plays an important role in temperature and pain sensation. In this project we aimed at discovering how the channel properties of TRPM3 are regulated at molecular and cellular level, and how these regulatory mechanisms can influence the channel’s versatile role in the sensory functions of the skin.Using a wide array of state of the art techniques, we discovered that a special subset of cell membrane lipids, phosphoinositols are necessary to the normal functioning of TRPM3: if the phosphoinositols are degrading, TRPM3 activity is decreasing. We also showed that external stimuli activating phospholipase C via other receptors on the cell surface also inhibited TRPM3, because phospholipase C degraded the phosphoinositols. In the other hand, ATP the central energy carrier molecule of the cells, (re)activated TRPM3 by fueling the re-synthesis of phosphoinositols. However, without being metabolized, the ATP itself directly inhibited TRPM3. In addition, we showed that inhibitors of protein kinase C, another important enzyme of cellular signaling pathways also inhibited TRPM3 suggesting a positive role of protein kinase C in the control of TRPM3. Selective overexpression of various isoforms of the enzyme revealed of differential role of the isoenzymes in the control of TRPM3. Moreover, we identified several chemical compounds directly influencing TRPM3 activity.Importantly, we also described a novel molecular gating mechanism of TRPM3, by opening an alternative ion permeation pore, which shows close similarities with the so called omega pore known from some mutant potassium and sodium channels. This was the first description of this mechanism on any native ion channel and it radically shaped our general knowledge about the activation properties of TRP channels. As we showed in animal experiments, this novel gating mechanism can 'short-circuit' TRPM3, thus aggravating the onset of pain. Altogether, these results provided a deeper insight in the cellular regulation and molecular interactions of TRPM3 which is essential to understand how this nociceptive ion channel can be involved in pathological conditions, like inflammation which are associated with altered sensory phenomena like hypersensitivity or allodynia. This fundamental knowledge can provide a solid base for further applied research activities especially in developing TRPM3 targeting drugs as potential analgesics. In control experiments, we accidentally discovered that the macrolide immunosuppressant rapamycin activated another thermosensitive TRP channel, the cold sensitive TRPM8. Since macrolide immunosuppressants are widely used in the clinical practice to manage various conditions and TRPM8 is a close relative of TRPM3 expressed in skin and innervating sensory neurons either, we further investigated this newly recognized pharmacological interaction which significantly extended the scope of our project. Studying the molecular interaction between various macrolides and TRPM8, we successfully identified the key motif of the compounds as well as amino acids residues of TRPM8 forming the potential binding site for the macrolides. We also showed, that rapamycin is more selective in activating cold sensitive TRPM8 expressing neurons than classical agonists, like menthol which activates TRPA1 either. Beyond macrolides, we also identified allyl isothoicyanate (AITC), the main pungent component of mustard oil as a TRPM8 activator. However, kinetic analysis and advanced modeling revealed distinct biophysical mechanisms: rapamycin, like classical TRPM8 agonists e.g. menthol, stabilized the open state of TRPM8 resulting in slower closing, while AITC destabilized the close state resulting in faster opening of the channel. Based on these kinetic differences we were able to predict by modeling and later validating experimentally, that classical agonist are more effective in activating sensory neurons then would be expected from recombinant systems. These results of the project provided a better understanding of pharmacological properties of TRP channels which may have an enormous impact on pharmaceutical design and later drug developmental projects. Moreover, we also identified TRPM8 as a new target of macrolide immunosuppressants which may reveal the possibility of novel therapeutic applications.Furthermore, we also investigated the role of further temperature sensitive TRP channels in the skin. In frame of an international collaboration led by the Laboratory of Cellular and Molecular Physiology at University of Debrecen, the previous host laboratory of the fellow, we studied TRPV4 another heat sensitive member of TRP channels mainly expressed by the epithelial cells of the skin. We found, that the cannabidiol, a non-psychoactive phytocannabinoid exert a strong anti-acne effect which is partially mediated by TRPV4 expressed on the cells of the sebaceous glands. These results have a direct clinical impact providing novel therapeutic approach in the treatment of acne vulgaris, a common inflammatory skin disease.During the execution of the project, we established new international collaborations with the Department of Physiology and Pathophysiology at University of Marburg (Marburg, Germany), and with the Department of Physiology at University of Debrecen (Debrecen, Hungary). These collaborations significantly contributed to the success of the project and resulted in an important broadening of the international network of the all participants. both laboratories and contributes to mutual research projects and joined publications. Moreover, the fellow was also active in “in house” collaborations in the KU Leuven. He was responsible for the electrophysiological characterization of induced pluripotent stem cell derived neurons provided by the Laboratory of Stem Cell Biology and Embryology in joint research projects which had a great added value to extend his electrophysiological expertise.Altogether, this project had an extraordinary contribution to the supported fellow’s career development. He became an expert in electrophysiology and familiar with animal experimentation. He acquired significant new knowledge about TRP channels and sensory physiology and developed important complementary skills nominating him for a successful career as independent, leading researcher. Furthermore, some as yet unpublished findings will form a firm basis for his future research projects. His return to his original host institution after this fellowship realize a significant knowledge transfer and long term synergies between the two laboratories further increasing the competitiveness of the European Research Area. He already did the first steps toward the successful transition: his first Hungarian basic research grant application is already under evaluation, in which he plans further characterize pharmacological interactions of TRPM3 to better exploit its drug developmental potencies. He also gained the highly prestigious Bolyai fellowship from the Hungarian Academy of Science further supporting his future carrier in Hungary.
