Illuminating the TRPC3 signaling machinery

Ion channels of the TRPC family are protein molecules (proteins) that are found in the cell membrane of almost all human cells and represent targets for the drug (pharmacological) treatment of various diseases. The name TRPC is derived from the function of these molecules in the insects where they were first discovered. A defect in the TRPC function leads to blindness in the fruit fly by shortening the reaction time of photoreceptors, which is known as the transient receptor potential (TRP) phenotype. Disturbances in the function of classical TRP molecules (TRPCs) are believed to be responsible for diseases of the nervous system (neurodegeneration), the cardiovascular system (cardiac remodeling and heart failure, cardiac arrhythmia) and the kidneys (renal failure), but also for tumor diseases. Accordingly, the development of drugs, which target TRPC channel proteins appears highly attractive for the development of new, improved therapies against these diseases. The central hurdle on the way to this development is the wide distribution of similar TRPC channels in the human body, which makes a high-specificity of the therapeutic intervention required for successful clinical application. Furthermore, it is still largely unclear whether and how certain TRPC channels in different organs of the human body differ in their function. A prominent feature of the TRPC3 protein, representing an important member of this ion channel family, is the ability to recognize its environment within the cell membrane with respect to its fat (lipid) composition. Special membrane lipids are bound to as yet not clearly identified sites within the TRPC3 molecule and thus determine its function. Recently, it has become possible to control the effect of such regulatory lipids or synthetic agents that bind to TRPC3 channels, specifically and effectively by light. The active state of regulatory lipids and also of synthetic agents can be switched on and off by light of specific wavelengths. Hence, light-mediated control of the TRPC3 function is possible without direct contact, i.e. only by light and with very high temporal and spatial precision. This method of light-mediated control of proteins and thus of organ functions is termed photopharmacology. In the present project, on the one hand, the exact molecular mechanisms of the influence of lipids and synthetic agents on TRPC3 are to be clarified and, on the other hand, strategies for a completely new and highly specific control of these molecules in diseased human tissues by photopharmacology are to be developed.

Project P 33263-B was designed to increase our knowledge about a Ca2+ permeable ion channel designated as TRPC3 (transient receptor potential channel canonical 3). This cation channel is essential for the generation of Ca2+ signals in the cells of a large variety of human tissues. TRPC3 has been proposed as a potential target for the therapy of neurological disorders such as epilepsy and cognitive dysfunction as well as neurodegeneration, but also for cardiovascular disorders, specifically for hypertension, pathologic cardiac remodeling and arrhythmia. Moreover, TRPC3 was indicated as a valuable target for the treatment of diabetes mellitus and some type of cancers. Despite an overwhelming body of evidence for a role of TRPC3 in human physiopathology, disappointingly little progress has to date been made towards therapeutic targeting of this ion channel. This shortcoming is in part caused by the lack of knowledge about the cellular control and specific regulatory structures within the TRPC3 protein. The working hypothesis for project P 33263-B builds on the concept that TRPC3 receives critical input from multiple lipids, which bind to regulatory sites within the channel complex. The overarching goal of this project was to advance the understanding of TRPC3 channels in terms of its regulation by lipids. To achieve these goal, we applied an interdisciplinary strategy that allowed for an efficient hypothesis-generation/hypothesis-testing cycle. This cycle started with computational modelling of lipid-channel interactions to identify candidate regulatory sites and critical residues within these regions of the protein. The relevance of these structures was tested by mutagenesis and electrophysiological recordings, and the functional phenotypes of TRPC3 mutations were then rigorously tested for consistency with the computational model. Moreover, the experimental strategy involved a novel approach for analyzing protein-lipid interactions. Active lipid species were introduced with high temporal and spatial precision using lipid photopharmacology. This allowed for the exposure of TRPC3 channels to well-defined levels of regulatory lipids. Overall, our work produced significant new insights into TRPC3 cellular regulation and function. Based on our results, we now propose TRPC3 as a lipid-sensitive channel that is endowed with the ability to recognize multiple regulatory lipids. We identified the mechanisms by which diacylglycerol, PIP2 and cholesterol control the activity of TRPC3 channels. Our work demonstrated that these lipids occupy distinct sites, which we currently designate as L1-L3. Moreover, we obtained evidence for allosteric communication between these lipid-sensing sites. We propose a concept in which complex lipid regulation adapts TRPC3 activity to the metabolic and functional state of tissues. Our findings pave the way towards a better understanding of the cellular role and the regulation of TRPC3 and are expected to promote the development of therapeutic interventions with TRPC3 as the target molecule.