Diplomarbeit Zur Erlangung des akademischen Grades Magister der Pharmazie an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz Characterization of a Novel Pharmacological TRPC3-Activator Eingereicht von Mohamad-Ali Baradaran Graz, Jänner 2016 Der experimentelle Teil der vorliegenden Arbeit wurde im Zeitraum von September 2014 bis Februar 2015 am Institut für Biophysik der Medizinischen Universität Graz durchgeführt. Ich möchte mich bei Prof. Dr. Klaus Groschner für die Themenstellung und die freundliche Betreuung, sowie für die Korrektur meiner Arbeit recht herzlich bedanken. Für die Einführung in die praktische Arbeitstechnik und als Ansprechpartner für Probleme während der Durchführung der Arbeit gebührt mein Dank Herrn Dr. Michael Poteser. Außerdem bedanke ich mich bei Frau Dr. Michaela Lichtenegger für die freundliche Hilfe im Laboralltag. Des Weiteren danke ich allen Mitarbeitern des Instituts für das angenehme Arbeitsklima. Zu guter Letzt danke ich meiner Familie für die jahrelange Unterstützung. 2 Table of Content 1) Introduction 4 1.1 TRP Channels 4 1.2 TRPC Channels 7 1.3 TRPC3 9 1.4 TRPC3-Activation 11 1.4.1 Activation-Mechanisms 11 1.4.2 Carbachol 16 1.4.3 GSK1702934A 16 1.5 Physiological / Pathophysiological Roles of TRPC3 17 1.5.1 Cardiovascular System 17 1.5.2 Nervous System 21 1.6 Pore structure and gating processes in TRPCs – the TRPC3G652A mutation 22 2) Aim 23 3) Material and Methods 25 3.1 Patch-Clamp Technique 25 3.2 Cell Culture 31 3.2.1 HEK-293 Cells 31 3.2.2 Cell Culture Performance 32 3.3 Solutions 33 3.4 Equipment and Analysis Software 37 4) Results 38 4.1 TRPC3 - Wildtype 38 4.1.1 Experiments in physiological extracellular Solution 38 4.1.2 Experiments with Ca2+ as the only extracellular charge carrier 42 4.2 TRPC3 - G652A 45 4.2.1 Experiments in physiological extracellular Solution 45 4.2.2 Experiments with Ca2+ as the only extracellular charge carrier 49 5) Discussion 52 5.1 Decreased lipid-sensitivity of TRPC3 by the G652A mutation 52 5.2 Pharmacological plasticity of the TRPC3 channel pore 53 5.3 Pharmacotherapeutic relevance of GSK1702934A 57 6) Abstract 59 7) Abbreviations 61 8) References 62 3 Introduction 1) Introduction 1.1 TRP Channels “Transient receptor potential” ( TRP )-proteins form a superfamily of non-selective cation channels which are mostly permeable for Ca2+ and monovalent cations. TRP channels seem to play important roles in many divergent physiological and pathophysiological processes, for example in the cardiovascular system, or in the nervous system. These Roles are not well understood yet, and because of that pharmacological strategies based on TRP channels as targets have so far not been developed. Therefore these channels are the subject of intensive scientific research. “The founding member of this superfamily was identified as a Drosophila gene product required for visual transduction, which in the fruit fly is a phospholipase C- dependent process” [ 1 ]. “The name transient receptor potential is based on the transient rather than sustained response to light of Drosophila flies carrying a mutant in the TRP locus” [2]. This TRP-mutant has a defect in light-induced Ca2+ influx [ 3 ]. Based on protein homology the TRP-Superfamily is subdivided into seven subfamilies: 1) TRPC “Classical” ( TRPC1-TRPC7 ); they show greatest similarity to Drosophila TRP. 2) TRPV “Vanilloid” ( TRPV1-TRPV6 ) 3) TRPM “Melastatin” ( TRPM1-TRPM8 ) 4) TRPA “Ankyrin” ( TRPA1 ) 5) TRPN „non mechanoreceptor potential C“ ( not expressed in humans ) 6) TRPP “Polycystin” ( TRPP2, TRPP3, TRPP5 ) 7) TRPML “Mucolipin” ( TRPML1-TRPML3 ) 4 Introduction Figure 1 : Transmembrane topology and phylogenetic tree of mammalian TRP channels “(A) Transmembrane topology (left) and the quartenary structure of TRP channels (right). The TRP protein has six putative transmembrane domains, a pore region between the fifth and sixth transmembrane domains and a TRP domain in the C-terminal region. The TRP protein assembles into homo-tetramers or hetero-tetramers to form channels. (B) Phylogenetic tree of mammalian TRP channels based on their homology.” [ 4 ]. 5 Introduction Figure 2 : Domain Structure of the TRP Superfamily [ 5 ] 6 Introduction 1.2 TRPC Channels The subfamily of the TRPC channels contains seven different non-selective cation channel isoforms ( TRPC1-TRPC7 ). The name “transient receptor potential classical” ( or “canonical” ) is because of the fact that within the superfamily of TRP-Channels, this subfamily shows the highest sequence similarity to Drosophila TRP-channel proteins. TRPC-Channels are non-selective cation channels which show permeability to Ca2+ and Na+-Ions. However, the permeability-ratio differs from channel to channel [ 6 ]. Due to structural and functional similarities these channels can be further subdivided into four groups: -TRPC1 -TRPC2 -TRPC3/6/7 -TRPC4/5 TRPC2, as a pseudogene is not expressed in humans [ 7 ]. 7 Introduction Figure 3: Expression and functions of TRPCs [ 8 ] 8 Introduction 1.3 TRPC3 TRPC3 Proteins form non-selective cation channels with predominant permeability for Ca2+ and Na+. TRPC3 Channels are expressed ubiquitously in both excitable and non-excitable cells, but their expression is predominant in specific regions of the brain and heart [ 9 ]. The understanding and picture of architecture of TRPC3 channels is still incomplete. The human TRPC3 protein consists of 848 amino acids with intracellular N- and C- termini [10]. However, different splicing variations of the TRPC3-gene have been identified. TRPC3 is supposed to be an integral membrane protein with seven membrane-spanning hydrophobic regions. Six of these hydrophobic domains form the transmembrane core domain ( TM1-TM6 ) [11]. The first hydrophobic region, is an intracellular, membrane-associated segment. The region between TM5 and TM6 is supposed to be the pore region of the channel. TRPC3 most likely forms tetrameric channel complexes. Figure 4: Topology diagram of single subunit of Pore region TRPC3 The N-terminus shows four ankyrin domains. These are followed by a linker and a coiled-coil domain. The C-terminus contains a coiled-coil domain, with the CIRB-Region ( CaM-IP3R-binding-site ) and the TRP-box. [12] 9 Introduction Figure 5: Cryo-reconstruction of TRPC3 [13] Figure 6: Molecular Structure of TRPC3: Side-view: Structural domains in TRPC3. One subunit is colored. The foremost TM1-TM4 module was removed for clarity. Top-view: Four subunits (chain A–D) forming the complete TRPC3 tetramer. [14] 10 Introduction 1.4 TRPC3-Activation 1.4.1 Activation-Mechanisms Physiologically, TRPC3 channels, which also show a significant basal activity [6], can apparently be activated through different ways. There is a common agreement that the channel is activated in response to receptor stimulation ( non-store-operated Ca2+-Entry , non-SOCE ) or through store depletion of intracellular Ca2+-stores ( store- operated Ca2+-Entry, SOCE ) [ 15 ]. Receptor stimulated Activation of TRPC3 Figure 7: Receptor stimulated Regulation of TRPC3-Acitivity. [16] 11 Introduction Stimulation of Gq-protein coupled receptors ( such as the mGluR1 receptor, the muscarinic acetylcholine receptors M1, M3, M5 , or the AT1 receptor for Angiotensin II ), as well as receptor tyrosine kinase membrane receptors ( such as the TrkB receptor ) leads to an activation of Phospholipase C ( PI-PLC ) which catalyzes the hydrolysis of phosphatidylinositol 4,5-bis-phosphate (PIP2) to generate inositol- 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Through interacting with the IP3- Receptor in the endoplasmatic reticulum ( ER ) membrane, IP3 causes a release of Ca2+ from the ER [ 15 ]. Diacylglycerol, a lipid mediator, accumulates in the plasmamembrane in the form of discrete droplets. These droplets localize within microdomains of the site of its origin [ 17 ]. The increase of the DAG concentration in the membrane is suggested to cause a “membrane curvature packing stress” which makes a physical stretch likely leading to channel activation [ 15 ]. PIP2 is needed for the activation of TRPC3 through DAG. The activation of TRPC3 leads to a Na+ and Ca2+ Influx into the cell. All in all, the activation of the PI-PLC Pathway results in a depletion of the intracellular Ca2+ stores and also in a Na+ and Ca2+ Influx from outside the Cell ending in an increase of the cytosolic Na+ and Ca2+ concentrations. This has various effects on cellular functions. For Example, it is supposed that the Na+ entry causes the depolarization necessary to activate voltage-operated (voltage- gated ) Ca2+ channels (VOC). It is also proposed that this Na+ entry causes a Ca2+ Uptake into the cell through the Na+/Ca2+ Exchanger ( NCX ) [ 16 ]. DAG also activates the Protein kinase C, which inhibits the TRPC3 channel directly through phosphorylation or through the Protein kinase G. How is this paradox effect of DAG explicable? It is possible that a compartmentalization of this signaling route exists, which could explain this functional property of DAG. A “lipid annulus” exists around many ion channels and transporters, such as the nicotinic acetylcholine receptor, and is essential for their proper function [ 18 ]. If such a lipid annulus exists around the TRPC3 Channel, it could be possible that DAG accumulation within this compartment would just activate the channel without reaching the protein kinase C, therefore it would not come to a PKC activation. With continuing accumulation of DAG, and after reaching a certain threshold it would be possible for DAG to