Characterization of cardiovascular TRPC3 channels

Over the past decade, evidence for a pivotal role of TRP (transient receptor potential) proteins in cellular Ca2+ signaling has accumulated, and the concept of TRP signaling complexes as a potential pharmacological target has emerged. TRPC proteins form cation channel complexes and are considered as highly versatile signal transduction molecules. Results obtained in heterologous expression systems suggest TRPC3 as a paradigm of multifunctional signal transduction. Due to its ability to interact with a variety of signaling partners, TRPC3 may serve cellular Ca2+ signaling by multiple mechanisms and may control a variety of distinct physiological functions. In the cardiovascular system, TRPC3 is a prominent TRPC species in endothelial cells and cardiac myocytes. The structure, regulation and functional role of cardiovascular TRPC3 channel complexes remain still elusive. Open questions that will be addressed in this project are: i) What are the pore-forming interaction partners of TRPC3 in native cardiovascular TRPC3 channel complexes, and what is the role of specific subunits as determinants of channel function? ii) What is the physiological significance of interactions between TRPC3 and cardiovascular signaling molecules such as caveolin-1 and NCX1 in terms of channel gating and/or transduction of input stimuli into distinct Ca2+ signals? These topics will be investigated using native macro- and microvascular endothelial cells as well as cardiac myocytes. The employed methods comprise classical techniques for subcellular localization of signaling molecules and for analysis of protein-protein interactions and subunit composition such as immunocytochemistry, immunoprecipitation and GST-pulldown experiments as well as analysis of mutation- induced changes in channel properties. These classical methods will be complemented by detection of protein- protein interactions within TRPC signalplexes with FRET- and single molecule-microscopy and by proteomic approaches to identify novel regulatory components and signaling partners of TRPC3. The function of cardiovascular TRPC signalplexes will be analyzed by measurement of membrane currents and intracellular ion concentrations. The proposed rigorous analysis of the molecular organization, function and physiological role of cardiovascular TRPC3 channels is suggested as an important step towards exploiting endothelial TRPC proteins as a therapeutic target.

Over the past decade, evidence for a pivotal role of TRP (transient receptor potential) proteins in cellular Ca2+ signaling has accumulated, and the concept of TRP signaling complexes as a potential pharmacological target has emerged. TRPC proteins form cation channel complexes and are considered as highly versatile signal transduction molecules. Results obtained in heterologous expression systems suggest TRPC3 as a paradigm of multifunctional signal transduction. Due to its ability to interact with a variety of signaling partners, TRPC3 may serve cellular Ca2+ signaling by multiple mechanisms and may control a variety of distinct physiological functions. In the cardiovascular system, TRPC3 is a prominent TRPC species in endothelial cells and cardiac myocytes. The structure, regulation and functional role of cardiovascular TRPC3 channel complexes remain still elusive. Open questions that will be addressed in this project are: i) What are the pore-forming interaction partners of TRPC3 in native cardiovascular TRPC3 channel complexes, and what is the role of specific subunits as determinants of channel function? ii) What is the physiological significance of interactions between TRPC3 and cardiovascular signaling molecules such as caveolin-1 and NCX1 in terms of channel gating and/or transduction of input stimuli into distinct Ca2+ signals? These topics will be investigated using native macro- and microvascular endothelial cells as well as cardiac myocytes. The employed methods comprise classical techniques for subcellular localization of signaling molecules and for analysis of protein-protein interactions and subunit composition such as immunocytochemistry, immunoprecipitation and GST-pulldown experiments as well as analysis of mutation- induced changes in channel properties. These classical methods will be complemented by detection of protein- protein interactions within TRPC signalplexes with FRET- and single molecule-microscopy and by proteomic approaches to identify novel regulatory components and signaling partners of TRPC3. The function of cardiovascular TRPC signalplexes will be analyzed by measurement of membrane currents and intracellular ion concentrations. The proposed rigorous analysis of the molecular organization, function and physiological role of cardiovascular TRPC3 channels is suggested as an important step towards exploiting endothelial TRPC proteins as a therapeutic target.