Characterization of a Novel Pharmacological TRPC3-Activator

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.

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

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 )

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 ].

Introduction

Figure 2 : Domain Structure of the TRP Superfamily [ 5 ]

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 ].

Introduction

Figure 3: Expression and functions of TRPCs [ 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]

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]

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 reach the PKC and activate it, ending into negative feedback for inhibiting the TRPC3 channel [ 15 ].

Introduction

Figure 8: Compartmentalization of the signaling route of the receptor stimulated TRPC3 activation:

A Open TRPC3 channel with hypothetical lipid annulus ( darker phospholipids ).

B PI-PLC generates Diacylglycerol which activates the TRPC3 Channel.

C After Increased DAG accumulation within the vicinity of the channel it is possible for DAG to reach the proteinkinase C and activate it. Proteinkinase C inhibits the channel through phosphorylation.

[ 15 ]

Introduction

Store-operated Activation of TRPC3

The role of TRPC3 in Store operated Calcium Entry ( SOCE ) into the Cell has been controversial [ 19; 20 ; 21 ]. TRPC3 has been associated with SOCE in different cell types, like pancreatic cells, prostate smooth muscle cells, HEK293 cells, hippocampal neuronal cells, and DRG neurons [ 22-26 ].

As already mentioned, the stimulation of Gq coupled receptors leads to an activation of the PLC Pathway ending into an increased production of inositol 1,4,5- trisphosphate (IP3). Through stimulation of the IP3 receptor in the ER membrane, IP3 causes an efflux of Ca2+ from the endoplasmatic reticulum.

This “depletion” of the intracellular Ca2+ stores is registered by STIM1 and STIM2, transmembrane proteins in the ER membrane, which function as Ca2+ sensors in the ER [ 27; 28 ].

The activation of STIM proteins leads to an aggregation with each other and a translocation to the plasmamembrane which ends in an interaction with calcium- selective Orai1 channels and TRPC channels. This was shown to promote opening of these channels, leading to an Ca2+ Influx into the cell, thereby increasing the cytosolic Ca2+ concentration and refilling the intracellular Ca2+ stores [ 26; 29].

Introduction

2+ Figure 9: Store operated Ca Entry ( SOCE )

“Model showing cellular events involved in activation of Orai1-mediated Ca2+ entry. Following depletion of the ER-Ca2+ stores, STIM1 aggregates and translocates to the ER–PM junctional regions where it binds to and activates the Orai1 channel. The Orai1-mediated Ca2+ entry may induce the trafficking of homomeric TRPC channels or heteromeric TRPC channels (e.g., TRPC1/TRPC3 and TRPC1/TRPC4) to the cell surface, following by insertion into the plasma membrane and subsequent gating by STIM1. Additionally, the extracellular Ca2+ entering via Orai1 may also directly activate other channels, such as TRPC5. ER, endoplasmic reticulum; PM, plasma membrane.“

[29]

Introduction

1.4.2 Carbachol

Carbachol ( 2-[(Aminocarbonyl)oxy]-N,N,N-trimethylethanaminium chloride ) is a direct parasympathomimetic, which stimulates muscarinic and nicotinic acetylcholine receptors. It is an acetylcholine analogon in which the acetyl-group is exchanged to a carbamoyl-group, giving the drug more resistance against hydrolysis through the Acetylcholinesterase.

Carbachol is used in ophthalmology in treatment of glaucoma and also in surgical procedures such as cataract extraction [30 ].

As a muscarinic receptor agonist it can also be used for pharmacological activation of

TRPC3 through stimulation of the Gq coupled muscarinic receptors M1, M3, and M5.

1.4.3 GSK1702934A ( = “GSK” )

GSK1702934A is a novel TRPC3-activator which was introduced recently [ 31 ]. This pharmacological TRPC3-agonist is in the focus of the present study.

Introduction

1.5 Physiological / Pathophysiological Roles of TRPC3

TRPC3 as a multifunctional molecule has been suggested to play relevant roles in different Physiological / Pathophysiological Processes. 1.5.1 Cardiovascular System

Heart failure ( HF ) is a pathophysiological condition in which the heart is not able to pump enough blood to keep up the blood flow, which is required to sustain physiological functions of the body [ 32 ]. The reduction of cardiac output and further the lack of the blood supply to organs, leads the body to initiate compensatory mechanisms. These are for example an increased secretion of adrenalin and noradrenalin through Sympathicus activation, which elevates the heart muscle's contractility, an increased activation of the Renin-Angiotensin-Aldosteron-System ( RAAS ), an increased secretion of adiuretin, and an increased Secretion of Endothelin-1. Although these mechanisms are necessary for temporary maintaining the blood supply of the organs, they will damage the heart muscle's proper function in the long run, leading to a hypertrophy of the heart muscle ( Hypertrophic Cardiomyopathy ) [ 33 ]. The “cardiac remodeling” leads to an increased stiffness of the heart muscle, which obstructs a sufficient diastolic blood filling of the heart.

“A critical step in the early stage in the cardiac remodeling is up-regulation of the expression of certain Ca2+ channel proteins to modify cardiac Ca2+ homeostasis and Ca2+ transcription coupling” [ 34 ].

The Expression of TRPC channels in the heart is up-regulated in response to mechanical stress or increased neurohumoural stimuli [ 35; 36; 37 ].

TRPC3 is important for the Ca2+ dependent regulation of calcineurin,[38; 39] an enzyme which plays an important role in the genexpression in cells, leading to cardiac hypertrophy [ 40; 41 ]. It has been shown that TRPC3 is essential for the angiotensin II-induced cardiac hypertrophy [ 42 ]. Based on this knowledge and currently available evidence, TRPC3 appears as an attractive pharmacological target for prevention of chronic heart failure development.

Introduction

Figure 10: Neurohormonal compensatory mechanisms in heart failure

The decreased cardiac output leads to a generation of afferent signals by high-pressure baroreceptors, which ends in an activation of compensatory mechanisms: Release of Adiuretin ( AVP ), Activation of efferent sympathetic nervous system, and activation of the Renin-Angiotensin- Aldosteron-System [43].

Introduction

Figure 11: Hypertrophic cardiomyopathy [ 44 ].

Cardiac arrhythmia is a condition in which the beat of the heart is irregular [33]. Too fast heartbeats ( more than 100 beats per minute in adults ) are called Tachycardia, and too slow heartbeats ( less than 60 beats per minute in adults ) are called Bradycardia. Cardiac arrhythmia can lead to sudden cardiac death [ 45 ].

Recent studies on TRPC3 overexpressing mouse hearts showed that TRPC3 contributes to arrhythmogenesis [ 34 ]. A functional partnership between TRPC3 and NCX1 has been proposed in special micro / nanodomains. NCX1 is an antiporter membrane protein which transports Ca2+ and Na+ ions over the cellmembrane. The antiporter transports three Na+ ions into the cell and one Ca2+ ion out of the cell with every circle ( “NCX forward mode” ). The driving force for this transport is the much higher extracellular Na+-concentration [ 46 ]. It is suggested that TRPC3 influences the operation mode of NCX1 by increasing the intracellular Na+ concentration. The increased Na+ level reduces the Ca2+ extrusion by the NCX1, ending even in a Ca2+ entry at positive potentials.

Introduction

This “Reverse mode NCX-mediated Ca2+ Entry”, along with the Ca2+ entry through TRPC3, results in an increased cytosolic Ca2+ Concentration with positive inotropic effects.

Excessive TRPC3 Activation leads to an inactivation of TRPC3 channel and a spatial uncoupling between TRPC3 and NCX1. “The rapid drop in local cytoplasmic Na+ at the exchanger is expected to transiently favour arrhythmogenesis by facilitation of NCX forward mode” [ 34 ]. The changed mode of the NCX is expected to promote abnormal depolarizations of cardiac myocytes.

Figure 12: “Hypothetical model of dynamic, functional interaction between TRPC3 and NCX1.” [ 34 ]

Introduction

Pulmonary Arterial Hypertension: Pulmonary arterial hypertension is an increase of the blood pressure in the pulmonary artery [ 86 ]. Idiopathic pulmonary arterial hypertension (IPAH) is a rare fatal disease that causes right heart failure and death [ 87 ]. The increased pulmonary vascular resistance is mainly caused by a vascular hypertrophy, which is a result of a massive proliferation of pulmonary artery smooth muscle cells ( PASMC ). Increased Ca2+ Influx is a very important stimulus for the proliferation of these cells. Studies showed an increased expression of TRPC3 and TRPC6 in lung tissues and PASMCs from IPAH patients, compared to those from normotensive or secondary pulmonary hypertension patients. This observation leads to the consideration that TRPC channel overexpression may be partially responsible for the excessive proliferation of PASMCs and pulmonary vascular hypertrophy in IPAH patients [ 87 ].

1.5.2 Nervous System

Central Nervous System: Brain-derived neurotrophic factor ( BDNF ) is an important Neurotrophin which occurs in the central nervous system, but also in peripher tissues [ 47; 48 ]. It has been shown that BDNF is important for the survival of existing Neurons, and also for the growth and differentiation of new neuronal cells and synapses [ 49; 50 ]. BDNF seems to play important roles in pathological conditions of the central nervous system like Alzheimer's disease, Epilepsy and Schizophrenia [ 51 ; 52; 53 ]. As recent studies showed a functional coupling between the TRPC3 channel and BDNF, TRPC3 may be a potential target in pharmacotherapy of these diseases [ 54; 55; 56 ].

Chronic Pain: Recent studies on rat dorsal root ganglion ( DRG ) neurons show that TRPC3 is functionally coupling to pro-inflammatory receptors which leads into hyperalgesia by sensitization of nociceptors, making TRPC3 an interesting potential target for pharmacological treatment of chronic pain [ 26 ].

Introduction

1.6 Pore structure and gating processes in TRPCs – the TRPC3G652A mutation

TRPC3G652A is a mutant of TRPC3 in which the amino acid glycine in position 652, in the transmembrane domain 6, the putative pore region of the channel, is exchanged with alanine, giving the mutant decreased lipid sensitivity [ 57 ].

( A )

( B )

Figure 13:

( A ) A model of the channel pore displayed as TRPC3 dimer based on TRPV1. Colour code: Blue – S5, orange – S6, deep cyan blue – pore loop [ 57 ].

( B ) Structural formulas of the amino acids glycine ( left ) and alanine ( right ).

Aim

2) Aim

“Ion channels have 2 fundamental properties, ion permeation and gating. Ion permeation describes the movement of ions through the open channel. The selective permeability of ion channels to specific ions is a basis of classification of ion channels (e.g., Na+, K+, and Ca2+ channels). Size, valence, and hydration energy are important determinants of selectivity. Gating is the mechanism of opening and closing of ion channels and is their second major property. Ion channels are also subclassified by their mechanism of gating: voltage-dependent, ligand-dependent, and mechano- sensitive gating” [ 66 ].

Gating in terms of activation and / or inactivation mechanisms of many different ion channels have been in the focus of many experimental studies in the last decade, but little is known about the possibility of a gating-associated modulation of the permeation properties of ion channels. “The selectivity of ion channel pores has generally been viewed as fixed” [ 80 ].

However, experimental studies on P2X-receptors and nicotinic acetylcholine receptors showed, that there is a possibility to modulate the channel's ion selectivity [ 67; 68; 69 ].

The aim of the present study was to characterize the impact of different TRPC3- activators on the channel's permeation properties. Therefore we compared the permeation properties of TRPC3 when activated by two different stimuli: the cholinergic agonist Carbachol and the novel TRPC3-agonist GSK1702934A ( “GSK” ).

Besides investigations with wild-type TRPC3 channels, we also intended to characterize permeation and gating as well a linkage between these functions in the G652A mutation.

Aim

Figure 14: Molecular elements of an ion channel

Material and Methods

3) Material and Methods 3.1 Patch-Clamp Technique

The Patch-Clamp Technique is a laboratory method in electrophysiology which allows the measurement of currents through biological membranes. It is used for the study of ion channels and electrogenic transporters in the cell membrane of non-excitable cells and excitable cells like neurons and muscle cells.

The Patch-Clamp Technique was developed by Bert Sakmann and Erwin Neher in the late 1970s and early 1980s. For this revolutionary development, which made it possible to measure currents through single ion channels for the first time, Sakmann and Neher received the Nobel Prize in Physiology or Medicine in 1991.

Setup: A glass capillary ( “patch pipette” ) filled with suitable solution ( “internal solution” ) contains the recording electrode. The reference electrode is in the bath solution ( “external solution” ) around the cell. The patch pipette is pressed against the cell membrane which results in an electrical isolation of a patch of membrane from the external solution ( “sealing” ). This set-up allows the measurement of electrical current flowing into the patch.

Figure 15: Diagram of the Patch-Clamp technique ( whole cell configuration ) [ 58 ].

Material and Methods

Depending on what is going to be studied different configurations are possible:

1) Cell-attached Configuration: Figure 16 [59] The patch pipette is pressed against the cell membrane, and after applying a slight suction, a “gigaseal”, with an electrical resistance of more than 1 Giga ohm between the interior of the pipette and the external solution, is formed. This configuration, in which the cell membrane remains intact, allows the measurement of currents through single ion channels. The advantage of this configuration on the one hand is that the cell's physiological mechanisms remain intact, allowing the study of their influence on the channel's activity. On the other hand it is not possible to modify the cell's internal mechanisms directly with this configuration.

2) Whole-cell Recording: After forming a gigaseal, the sealed membrane patch is ruptured by a pulse of voltage or suction, connecting the pipette's interior with the cytosol of the cell. This configuration allows the measurement of currents through entire cell membranes. The advantage of this configuration is that, it allows a direct modulation of the cell's interior with the internal solution. This configuration was used for the present study.

Figure 17 [ 60 ] 26

Material and Methods

3) Inside-out: Starting from the cell-attached mode, this configuration is achievable by lifting the pipette from the cell membrane, resulting in a detached membrane patch on the pipette's tip. In this configuration the cytosolic surface of the membrane patch is exposed to the external solution. Therefore, it is possible to modulate the channel's interior side with compounds in the external solution.

Figure 18 [ 61 ]

Material and Methods

4) Outside-out: Starting from the whole-cell mode, this configuration is reached by lifting the pipette off the cell membrane. The detached cell membrane fragments reform into a lipid bilayer on the tip of the pipette, with the former extracellular side of the membrane facing the external solution. In this configuration it is possible to modulate the channel's interior side with the pipette solution. It is also possible to expose the extracellular surface of the membrane to different external solutions one after another.

Figure 19 [ 62 ]

Material and Methods

Figure 20 [ 63 ] 5) Perforated patch: After forming the giga ohm seal in the cell-attached mode, special compounds in the internal solution form small pores in the membrane patch, resulting in a configuration which is very similar to the whole-cell mode. The perforated patch provides electrical access to the cell's interior, but allows just restricted exchange between the cytosol and the pipette solution. Therefore, most of the intracellular mechanisms and also their effects on channel's activity are preserved. Furthermore, this configuration is usually more stable than the whole-cell mode.

Figure 21 [ 64 ] 6) Loose patch: In this configuration the pipette is moved towards the cell until the electrical resistance is a few times greater than that of the electrode alone. Without forming a connection between the pipette and the cell membrane, there is still a “leak” left between the cell and the pipette tip. The advantage of this configuration on the one hand is the possibility to make repeated measurements on the same cell. But on the other hand the leak between the cell and the pipette causes a “leak current” which negatively influences the measurements. However, this leak current can be partially corrected in some cases.

Material and Methods

Measurement: In general the Patch-Clamp technique can be used for two different kinds of measurements:

1) Current-Clamp: In the current clamp mode, an applied current is kept constant, while measuring the changes in the membrane voltage.

This method is used for example for the measurement of action potentials in neurons and muscle cells.

2) Voltage-Clamp: In the voltage clamp mode, which was used for the present study, a controlled voltage ramp is applied to the cell, while measuring the current through a single channel or the whole cell membrane.

The whole-cell voltage clamp mode was used in all measurements in the present study.

The holding potential was 0 mV, and the voltage ramp ranged from -130 mV to +80 mV.

Material and Methods

3.2 Cell Culture

3.2.1 HEK-293 Cells

Human Embryonic Kidney 293 Cells ( “HEK cells” ) are a specific cell line of originally human embryonic kidney cells transformed with human adenovirus 5 DNA. This cell line was generated in 1973 in Alex J. van der Eb's laboratory at the University of Leiden in the Netherlands. The Transformation of these cells was performed by Frank Graham and the number 293 comes from the fact that this was Frank Graham's 293rd experiment.

Since HEK cells are relatively easy to cultivate and to transfect, they are widely used in cell biology for analyzing overexpressed proteins since many years. HEK cells are epithelial cells with adherent culture properties and biosafety level 2. They endogenously express the muscarinic receptors M1, M3, and M5 [ 70 ].

Figure 22: Human Embryonic Kidney 293 Cells [ 65 ] 31

Material and Methods

3.2.2 Cell Culture Performance

This study used HEK-293 cells ( ATCC ) stably transfected with wild-type TRPC3 or the mutant TRPC3G652A.

Cultivation of the Cells: The Cells were grown at a temperature of 37°C, a CO2-level of 5% and a relative humidity of 80%. They were splitted every second day: After removing the medium, the cells were washed with PBS-buffer and incubated with trypsin solution for 3 minutes at 37°C. The effect of trypsin was stopped with diluting the received cell suspension with DMEM culture medium. This cell suspension was then centrifuged for 3 min at 1500 rpm. After removing the supernatant, the cell pellet was resuspended in DMEM culture medium containing Geneticin ( “G418” ; final concentration: 1 μg/ml ) as a selection antibiotic.

Preparing the Cells for Measurement:

Glass coverslips were exposed to Poly-L-Lysine hydrobromide for 15 minutes to improve the cell adhesion. The cells were harvested as described above and seeded on the coverslips. Cells were incubated for at least 8h to allow them to settle down. Subsequently, they were subjected to patch clamp experiments.

The TRPC3Wildtype / TRPC3G652A -protein was marked with Yellow Fluorescent Protein ( YFP ). The expression and localization of the fusion-protein was verified via fluorescence microscopy at 488 nm.

Figure 23: YFP-marked TRPC3 overexpressing HEK cells at 488 nm

Material and Methods

3.3 Solutions

All solutions were preheated to 37°C in a water bath before use.

Culture Medium DMEM 500 mL

Pen Strep 5,5 mL

L-Glutamine 5,5 mL

Hepes Buffer 5,5 mL

FBS 50 mL

Dulbecco's Modified Eagle Medium ( DMEM ): Sigma-Aldrich; D6429

Pen Strep: 5,5 ml of solution with 10,000 U/mL; Gibco Life technologies; Ref.: 15140-163; Lot.: 1546524

L-Glutamine: 5,5 ml of solution with 200 mmol / L; SIGMA Life Science; G7513; Lot.: RNBD4216

Hepes: 5,5 ml of 1M solution; PAN Biotech; Cat. No.: P05-01100; Lot No.: 1780714

Fetal Bovine Serum ( FBS ): 10% in final culture medium. SIGMA Life Science; F7524; Lot.: 074M3264

Phosphate Buffered Saline ( PBS ) NaCl 137 mM

KCl 2,7 mM

Na2HPO4x2H2O 8 mM

KHPO4 1,5 mM

PBS: pH 7,4; 290 mOsmol/Kg; without calcium chloride; without magnesium chloride; Life Technologies; 10010-056;

Material and Methods

Trypsin / PBS - Solution Trypsin 0,025% PBS

Trypsin: Trypsin-EDTA-Solution was used: 5.0 g porcine trypsin and 2 g EDTA • 4Na per liter of 0.9% sodium chloride; SIGMA Life Science; T4174-100ML

Extracellular Solutions:

ECS 1 ( Extracellular Solution 1 ) pH 7,5 NaCl 145 mM

Hepes 10 mM

MgCl2 Hexahydrate 2 mM

Glucose 10 mM

CaCl2 Dihydrate 2 mM

ECS 2 ( Extracellular Solution 2 ) pH 7,4 NMDG ( N-Methy-D-glucamin ) 132 mM

Hepes 10 mM

Glucose 10 mM

MgCl2 Hexahydrate 2 mM

CaCl2 Dihydrate 3 mM

Ca-gluconat 7 mM

Material and Methods

Intracellular Solutions ( Pipette Solutions ):

ICS 1 ( Intracellular Solution 1 ) pH 7,3 Cesium Methanesulfonate 120 mM

Cesium Chloride 20 mM

MgCl2 Hexahydrate 5 mM

Hepes 15 mM

EGTA 3 mM

ICS 2 ( Intracellular Solution 2 ) pH 7,3 Cesium Methanesulfonate 140 mM

Hepes 15 mM

MgCl2 Hexahydrate 5 mM

BAPTA 10 mM

Solution to enhance cell adhesion to glass coverslips:

Poly-L-Lysine-hydrobromide

Poly-L-Lysine-hydrobromide 5 mg

H2O sterile 50 ml

Poly-L-Lysine-hydrobromide: SIGMA Life Science; P5899-5MG; Lot.: SLBB8053V

Material and Methods

Solutions for TRPC3-activation:

Carbachol Carbachol 100 μM ECS 1 / ECS 2

Carbachol: 100 μM; solved in ECS 1 / ECS 2; Alfa Aesar Company; L06674; Lot.: 10176054

GSK GSK 1 μM / 10 μM ECS 1 / ECS 2

GSK : synthesized by Thoma Glasnov at the Institute of Chemistry at the University of Graz.

For a final bath concentration of 1 µM or 10 µM, a 10 mM stock solution of GSK in DMSO was diluted with ECS 1 / ECS 2 as required.

Material and Methods

3.4 Equipment and Analysis Software

Following Equipment / Software was used for the present study:

Equipment:

-Micropipette Puller: Flaming / Brown Micropipette Puller MODEL P-1000, SUTTER INSTRUMENT USA

The pipettes were pulled from thin-wall-filament glass capillaries ( GC 150 TF-7.5; Harvard Apparatus, Edenbridge, UK )

-Microscope: OLYMPUS IX70 Fluorescence Microscope

-Amplifier: Axopatch 200B, Axon CNS Molecular Devices

-Digitizer: Digidata 1440A, Axon CNS Molecular Devices

Software:

-Clampex 10.2 for recording

-Clampfit 10.4 for analyzing data

-SigmaPlot 11.0 for graphing the Clampex data

Results 4) Results 4.1 TRPC3 - Wildtype

4.1.1 Experiments in physiological extracellular Solution

For measurements under “Standard conditions” the patch pipette was filled with the “Intracellular Solution 1” ( ICS 1 ). The “Extracellular Solution 1” (ECS 1) was used as bath solution. Note that ICS 1 contained Caesium as the main charge carrier.

TRPC3-Wildtype showed a slight basal activity, which was increased through activation with carbachol 100 μM, GSK 1 μM, or GSK 10 μM ( Figures 24 and 25 ). However, the increase of current through GSK-activation (1 μM and 10 μM ) was much bigger than the activation current through carbachol. Raising the GSK-concentration from 1 μM to 10 μM increased the response slightly ( Figure 25 ).

Time course of current shows, that the response was rapid and transient in all cases.

GSK, in both concentrations, caused a much stronger influx than carbachol. This is shown very well in the time course of current ( Figure 26 ).

Compared to carbachol, GSK caused a shift in the reversal potential to more negative potentials ( Figure 24 ).

Results

Figure 24:

(A) I/V –plot of TRPC3 wildtype current derived from stimulation with carbachol 100 µM. Shown is the control current immediately before stimulation ( Control ) and maximum carbachol stimulated current

( Cch max. ). The reversal potential of 19,42 mV ( SEM=3,69; N=6 ) is marked by the arrow ( ER ). (B) I/V –plot of TRPC3 wildtype current derived from stimulation with GSK 1 µM. Shown is the control current immediately before stimulation ( Control ) and maximum GSK stimulated current ( GSK max. ). The reversal potential of 3,49 mV ( SEM=2,49; N=5 ) is marked by the arrow ( ER ). (C) Current density ( pA / pF ) at -80 mV through TRPC3 wildtype. Carbachol 100 µM induces a current density of -1,75 ( SEM=0,28; N=6 ); GSK 1 µM a current density of -50,29 ( SEM=12,48; N=5); GSK 10 µM a current density of -50,60 ( SEM=10,63; N=4).

(D) Reversal potentials ( mV ) of TRPC3 wildtype currents derived from stimulation with carbachol 100 µM, GSK 1 µM and GSK 10 µM. Carbachol 100 µM causes a reversal potential of 19,42 mV ( SEM=3,69; N=6 ); GSK 1 µM a reversal potential of 3,49 mV ( SEM=2,49; N=5 ); GSK 10 µM a reversal potential of 4,47 mV ( SEM=1,23; N=4 ).

Data are given as the means ± SEM. Statistical significance was determined by Student's t-test for unpaired values: * P ≤ 0,05

Results

Figure 25:

I/V –plot of TRPC3 wildtype current derived from stimulation with GSK 10 µM. Shown is the control current immediately before stimulation ( Control ) and maximum GSK 10 µM stimulated current ( GSK 10 µM max. ). The GSK 1 µM stimulated current maximum is also shown for comparison ( GSK 1 µM max. ). The reversal potential of 4,47 mV ( mean; SEM=1,23; N=4 ) is marked by the arrow

( ER ).

Results

Figure 26:

(A) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 wildtype derived from stimulation with carbachol 100 µM. (B) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 wildtype derived from stimulation with GSK 1 µM. (C) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 wildtype derived from stimulation with GSK 10 µM.

Results 4.1.2 Experiments with Ca2+ as the only extracellular charge carrier

For measurements under these conditions the patch pipette was filled with “Intracellular Solution 2” ( ICS 2 ). The “Extracellular Solution 2” ( ECS 2 ) was used as bath solution. Note that ICS 2 was buffered with BAPTA to avoid any disturbance due to elevation of intracellular Ca2+.

TRPC3-Wildtype showed a slight basal activity, which was increased through activation with carbachol 100 μM or GSK 1 μM. However, the increase of current through GSK-activation was much bigger than the activation current through carbachol ( Figure 27 ).

Time course of current shows, that the response was rapid and transient in all cases but it seems that the inactivation of the channel is a bit slower due to the buffering of the intracellular Ca2+ with BAPTA ( Figure 28 ).

Compared to carbachol, GSK caused a shift in the reversal potential to more negative potentials ( Figure 27 ).

Under these conditions the GSK currents were much lower than the currents in the experiments in physiological extracellular solution ( compare Figures 24 and 27 ).

Results

Figure 27:

( Cch max. ). The reversal potential of 1,05 mV ( SEM=7,30; N=7 ) is marked by the arrow ( ER ). (B) I/V –plot of TRPC3 wildtype current derived from stimulation with GSK 1 µM. Shown is the control current immediately before stimulation ( Control ) and maximum GSK stimulated current ( GSK max. ). The reversal potential of -12,89 mV ( SEM=1,86; N=12 ) is marked by the arrow ( ER ). (C) Current density ( pA / pF ) at -80 mV through TRPC3 wildtype. Carbachol 100 µM induces a current density of -1,66 ( SEM=0,49; N=7 ); GSK 1 µM a current density of -4,90 ( SEM=1,37; N=12 ). (D) Reversal potentials ( mV ) of TRPC3 wildtype currents derived from stimulation with carbachol 100 µM and GSK 1 µM. Carbachol 100 µM causes a reversal potential of 1,05 mV ( SEM=7,30; N=7 ); GSK 1 µM a reversal potential of -12,89 mV ( SEM=1,86; N=12 ).

Data are given as the means ± SEM. Statistical significance was determined by Student's t-test for unpaired values: * P ≤ 0,05

Results

Figure 28: (A) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 wildtype derived from stimulation with carbachol 100 µM. (B) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 wildtype derived from stimulation with GSK 1 µM.

Results 4.2 TRPC3 - G652A

4.2.1 Experiments in physiological extracellular Solution

TRPC3 G652A showed a slight basal activity, which was increased through activation with carbachol 100 μM, GSK 1 μM, or GSK 10 μM (Figures 29 and 30 ). However, the increase of current through GSK-activation (1 μM and 10 μM ) was much bigger than the activation current through carbachol. Raising the GSK-concentration from 1 μM to 10 μM increased the response slightly ( Figure 30 ).

Time course of current shows, that the response was rapid and transient in all cases.

GSK, in both concentrations, caused a much stronger influx than carbachol. This is shown very well in the time course of current ( Figure 31 ).

Compared to carbachol, GSK caused a shift in the reversal potential to more negative potentials ( Figure 29 ).

All currents ( means ) derived threw activation of TRPC3 G652A were lower than those derived from TRPC3 Wildtype activation ( Figure 24C and Figure 29C ).

Results

Figure 29:

(A) I/V –plot of TRPC3 G652A current derived from stimulation with carbachol 100 µM. Shown is the control current immediately before stimulation ( Control ) and maximum carbachol stimulated current ( Cch max. ). The reversal potential of 13,63 mV is marked by the arrow ( ER ).

(B) I/V –plot of TRPC3 G652A current derived from stimulation with GSK 1 µM. Shown is the control current immediately before stimulation ( Control ) and maximum GSK stimulated current ( GSK max. ).

The reversal potential of -2,23 mV ( SEM=5,65; N=3 ) is marked by the arrow ( ER ). (C) Current density ( pA / pF ) at -80 mV through TRPC3 G652A. Carbachol 100 µM induces a current density of -0,36 ( SEM=0,13; N=3 ); GSK 1 µM a current density of -43,10 ( SEM=14,45; N=4 ); GSK 10 µM a current density of -47,36 ( SEM=24,15; N=4 ). (D) Reversal potentials ( mV ) of TRPC3 G652A currents derived from stimulation with carbachol 100 µM, GSK 1 µM and GSK 10 µM. Carbachol 100 µM causes a reversal potential of 13,63 mV ( N=1 ); GSK 1 µM a reversal potential of -2,23 mV ( SEM=5,65; N=3 ); GSK 10 µM a reversal potential of 2,96 mV ( SEM=2,81; N=3 ). Data are given as the means ± SEM. Statistical significance was determined by Student's t-test for unpaired values: * P ≤ 0,05

Results

Figure 30: I/V –plot of TRPC3 G652A current derived from stimulation with GSK 10 µM. Shown is the control current immediately before stimulation ( Control ) and maximum GSK 10 µM stimulated current ( GSK 10 µM max. ). The GSK 1 µM stimulated current maximum is also shown for comparison ( GSK 1 µM max. ). The reversal potential of 2,96 mV ( mean; SEM=2,81; N=3) is marked by the arrow

( ER ).

Results

Figure 31: (A) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 G652A derived from stimulation with carbachol 100 µM. (B) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 G652A derived from stimulation with GSK 1 µM.

(C) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 G652A derived from stimulation with GSK 10 µM.

Results 4.2.2 Experiments with Ca2+ as the only extracellular charge carrier

TRPC3-G652A showed a slight basal activity, which was increased through activation with carbachol 100 μM or GSK 1 μM. However, the increase of current through GSK-activation was much bigger than the activation current through carbachol ( Figure 32 ).

Compared to carbachol, GSK caused a shift in the reversal potential to more negative potentials ( Figure 32 ).

Under these conditions the GSK currents were much lower than the currents in the experiments in physiological extracellular solution ( Figures 29 and 32 ).

All currents ( means ) derived threw charbachol induced activation of TRPC3 G652A were lower than those derived from TRPC3 Wildtype activation ( Figures 27C and 32C ).

Results

Figure 32:

The reversal potential of -10,3 mV ( SEM=2,15; N=6 ) is marked by the arrow ( ER ). (C) Current density ( pA / pF ) at -80 mV through TRPC3 G652A. Carbachol 100 µM induces a current density of -1,25 ( SEM=0,68; N=8 ); GSK 1 µM a current density of -4,94 ( SEM=1,91; N=6 ).

(D) Reversal potentials ( mV ) of TRPC3 G652A currents derived from stimulation with carbachol 100 µM, and GSK 1 µM. Carbachol 100 µM causes a reversal potential of 8,62 mV ( SEM=4,47; N=7 ); GSK 1 µM a reversal potential of -10,3 mV ( SEM=2,15; N=6 ). Data are given as the means ± SEM. Statistical significance was determined by Student's t-test for unpaired values: * P ≤ 0,05

Results

Figure 33: (A) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 G652A derived from stimulation with carbachol 100 µM.

(B) Time course of inward current ( at -80 mV ) and outward current ( at +70 mV ) through TRPC3 G652A derived from stimulation with GSK 1 µM.

Discussion

5) Discussion 5.1 Decreased lipid-sensitivity of TRPC3 by the G652A mutation

Gating of ion channels is understood as moving of the innermost transmembrane helices to open the permeation pathway [ 71 ]. This motion, very often requires a point of high flexibility called “hinge point” that is typically formed by the amino acids glycine or proline. Various studies have addressed the functional importance of “proline kinks” and putative “glycine hinges” in helices of ion channels [ 72-79 ].

The amino acid glycine in postion 652, in the transmembrane region 6, is supposed to be an essential structural element for the lipid-mediated gating of TRPC3 [ 57 ]. At this position, in the assumed pore region of the channel, we exchanged glycine with alanine through point mutation, and increased the side chain size of the amino acid. Note that the introduced amino acid alanine is uncharged and exhibits a small volume side chain. Therefore this mutation is expected to influence only gating properties of the channel and not the architecture and function of its selectivity filter.

Carbachol, as a muscarinic receptor agonist, stimulates the intracellular synthesis of DAG through the PI-PLC pathway, which, as lipid, activates the TRPC3 channel ( Chapter 1.4.1 TRPC3 Activation-Mechanisms ).

In our study carbachol-induced current density through the G652A mutant was much lower compared to TRPC3 Wildtype, whereas the GSK-induced current densities did not differ markedly ( Figures 24; 27; 29; 32 ).

Other experiments showed a lack of response of the G652A mutant to OAG (1-oleoyl- 2-acetyl-sn-glycerol ), another lipid TRPC3 activator [ 57 ].

These results show that the exchange of glycine with alanine in position 652 leads to a decreased lipid-sensitvity of the channel, making G652 a decisive point for the lipid- sensitivity and therefore lipid-mediated gating of TRPC3.

Discussion

5.2 Pharmacological plasticity of the TRPC3 channel pore

The selectivity of ion channels is their ability to discriminate among different ions. This ion selectivity is essential for their physiological function, and therefore for the physiological electrochemical gradient of ions over the cell membrane, which is necessary for the viability of cells. Without ion selectivity the production and conduction of electrical signals in biology would be impossible.

Ion channels adopt different molecular mechanisms to recognize and distinguish ions:

Firstly, ion channels have a charge selectivity. That means that they discriminate between cations and anions so that they are permeable either for cations or anions. This is due to charged amino acids in the pore region of them. The nicotinic acetylcholine receptor, a cation-selective ion channel, for example has negatively charged amino acid residues at the pore entrance, which don't allow anions to permeate the channel due to electrical repulsion [ 81 ].

Secondly, ion channels have selectivity filters which discriminate among different types of cations / anions. These selectivity filters usually differentiate ions through the filter diameter which allows only those ions to permeate, which have the suitable size to pass through. The selectivity filter of the voltage-gated sodium channel, for example, has a configuration, which selects ions by their size. That means that ions with a too big diameter are incapable to permeate the channel. The diameter of the channel pore is so narrow that just hydrated sodium ions are allowed to permeate, whereas other hydrated bigger cations like potassium ions are rejected [ 82 ]. Another selection mechanism is also the ability of ion channels to stabilize permeating ions within the structures of the selectivity filter. Voltage-gated potassium channels for example remove the hydration shell of potassium ions when they enter the selectivity filter. Carbonyl groups in the TVGYG-Sequence ( Thr-Val- Gly-Tyr-Gly ) in the selectivity filter interact with the dehydrated potassium ions and compensate their energy loss which provides an energetically favorable route for dehydration and therefore channel permeation of potassium ions.

Discussion

The structural configuration of the TVGYG-sequence and the diameter of the selectivity filter allow just dehydrated potassium ions to permeate, whereas smaller dehydrated sodium ions are not stabilized within the channel's filter, and are hence not able to permeate through these channels [ 82 ].

As mentioned above, the pore diameter of an ion channel is in principle a very important determinant of its ion selectivity. That means that the bigger the pore diameter is the less is the ion selectivity of the channel [83].

Experiments on acid-sensing ion channels ( ASICs ), which are voltage-insensitive cationic channels, show that they change their pore diameter at different pH, resulting in a different ion selectivity of these channels. At pH 7,25 ASIC1a showed a pore diameter of 10 Å with non selective currents. At pH 5,5 however, the pore diameter was much smaller with approximately 5 by 7 Å, allowing just the small sodium ions to permeate, ending in Na+-selective currents [ 84 ].

One measure, which provides information about the contribution of different ions in ion channel-mediated currents, is the Reversal Potential. In a biological membrane with opened ion channels the reversal potential is the membrane potential at which there is no current flowing through the membrane. The reversal potential consists of the equilibrium potentials of all ions which are capable to permeate the membrane's ion channels [ 85 ]. A change in the reversal potential is indicative of a change in the ion selectivity of the membrane's ion channels.

In the present study all measurements were done in TRPC3 wildtype / TRPC3 G652A overexpressing HEK-cells in the whole-cell voltage clamp configuration. That means the measured reversal potentials mainly consist of the equilibrium potentials of all ions in the used solutions which are able to permeate TRPC3 wildtype / TRPC3 G652A channels in the cell membrane.

The measurements were carried out on two different conditions with different intracellular and extracellular solutions, respectively ( See chapter 3.3 Solutions for the exact composition of the solutions ).

Discussion

Condition 1: Experiments in physiological extracellular solution.

Here we used ECS 1 and ICS 1 as the extracellular and intracellular solutions. The only ions in the solutions, which are known to permeate through TRPC3 are sodium, calcium, and cesium. Cs+ is the biggest cation among them, followed by Ca2+ and Na+ [ 89 ]. Note that the concentration of the physiologically relevant potassium ion inside the cells is too low to influence the measurements. So, if we activate the channel in the voltage-clamp configuration, we can see an influx-current into the cell and also an efflux-current out of the cell. Because of the composition of the used solutions we can assume that the influx under these conditions is most likely a sodium and calcium influx. The efflux however is most likely a cesium current.

Compared to carbachol, GSK-induced currents display a shift of the reversal potential to more negative potentials with both TRPC3 wildtype as well as TRPC3 G652A ( Figures 24 and 29 ).

Condition 2: Experiments with Ca2+ as the only extracellular charge carrier.

Here we used ECS 2 and ICS 2 as the extracellular and intracellular solutions. In these solutions Ca2+ and Cs+ are the only charge carriers to permeate through TRPC3. That means the influx is a Ca2+-current and the efflux a Cs+-current, respectively.

Under these conditions GSK-induced currents were characterized by an even bigger negative shift of the reversal potential as compared to Cch-induced currents with both TRPC3 wildtype and TRPC3 G652A ( Figures 27 and 32 ).

The result of this GSK-induced negative shift of the reversal potential is apparently reflecting a change of the permeability ratio of the channel in terms of larger cesium- derived efflux-current compared to the influx, which leads us to assume a higher conductance for cesium. In explanation of this change in the permeability ratio and further under the consideration that Cs+ was the biggest ion in all our experiments, we speculate that GSK induced a “pore dilation” in TRPC3 wildtype as well in TRPC3 G652A, which results in a decreased Ca2+ / Na+ -selectivity and an increased conductance for larger cations like Cs+.

Discussion

To our knowledge this is the first report of such an effect in TRPC3 wildtype / TRPC3 G652A mediated currents, but changes in the pore properties following agonist stimulation have been described for other members of the TRP channel family: TRPV1, TRPV3, and TRPVA1 [ 91-93 ].

An experimental study from 2009 on TRPA1 channels, which are believed to be mechanical and chemical stress sensors [94], shows that agonist stimulation of TRPA1 can induce the appearance of a large pore permeable to large organic cations such as N-methyl-D-glucamine ( NMDG ) [ 96 ]. This study suggests that the pore properties of TRPA1 and related TRPs are highly dynamic, and that native TRP channels are capable of entering the dilated state even in the presence of normal extracellular calcium concentrations, making this process a potentially important aspect of TRPA1 signaling. In this study it is speculated that “pore dilation” could be an important temporal aspect of TRPA1 signaling, which provides a mechanism for transient signal amplification during continuous exposure to potentially harmful chemicals.

Further, the group of Man-Kyo Chung and Ali D Güler showed that TRPV1, an ion channel which is highly expressed in sensory neurons and is involved in pain perception, shows dynamic ionic selectivity during agonist stimulation [ 95 ]. They found that the TRPV1 selectivity changes as a function of time, agonist concentration, ionic conditions, and even which agonist is applied. This change in the selectivity directly involves the TRPV1 channel pore, making the composition of the ions which permeate the channel an important qualitative signaling property of the channel.

As for TRPC3 of course more investigations have to be done to explore the physiological / pathophysiological roles of the potential TRPC3 channel pore plasticity.

Discussion

5.3 Pharmacotherapeutic relevance of GSK1702934A

At the end of the present study we want to make some considerations about the potential pharmacotherapeutic significance of our experimental observations. As mentioned above TRPC3 is known to be involved in many different pathophysiological conditions ( Chapter 1.5 Physiological / Pathophysiological Roles of TRPC3 ). These roles are too divergent to be all discussed here, but we want to focus on two diseases: The chronic heart failure ( CHF ) and the Idiopathic pulmonary arterial hypertension (IPAH). In both cases an overexpression of TRPC3 channels is known to, at least partially, cause a muscular hypertrophy of cardiac muscle cells / pulmonary artery smooth muscle cells, through an increased Ca2+ - Influx. This Ca2+ - induced muscular hypertrophy is a key pathological mechanism in the worsening of the patient's symptoms, and would hence be an interesting target in potential therapeutic strategies.

Based on this knowledge, a logical pharmacotherapeutic strategy in the treatment of these diseases would be to stop the excessive Ca2+ - Influx into cardiac muscle cells / pulmonary artery smooth muscle cells through an inhibition of TRPC3 channels with TRPC3 selective inhibitors. However, useful blockers with sufficient selectivity for TRPC3 channels are still missing. Such non-selective inhibitors are for example the organoborane, 2-aminoethoxydiphenyl borate ( 2APB ), and the imidazole derivate SKF96365 [ 88 ]. Other inhibitors are members of a class of pyrazole derivates called Bis-(trifluoromethyl)-pyrazoles ( PYRs ), like Pyr3 and Pyr10. Experimental studies showed that Pyr3, a previously suggested selective inhibitor of TRPC3, also inhibits Orai1-mediated Ca2+ Entry, whereas the novel pyrazole Pyr10 displayed significant selectivity for TRPC3 [ 90 ]. However, its selectivity to TRPC3 and its consequently potential pharmacotherapeutic significance has to be investigated in further studies.

GSK1702934A itself, as a TRPC3 channel activator, is not supposed to have benefits as a drug within a pharmacotherapy of these diseases.

Discussion

Due to our experiments another theoretical strategy would be to take advantage of the potential plasticity of the TRPC3 channel pore, in terms of using pharmacological channel modulators or partial agonists / antagonists which reduce the Ca2+ entry through “pore dilation”.

It is also possible that this “pore dilation” process changes the structural conformation of the channel to unmask new potential binding sites for TRPC3- blockers / modulators which are usually hidden within the protein. Such potential binding sites could be interesting targets for new TRPC3- modulators. This idea however, needs further investigation about the TRPC3 channel's pore structure in human tissues like cardiac muscle cells / pulmonary artery smooth muscle cells.

Abstract

6) Abstract

TRPC3 is a member of the TRP-channel superfamily, which forms non-selective cation channels with significant permeability for Ca2+ and Na+. TRPC3 is supposed to play important roles in physiological / pathophysiological processes and seems to be an interesting potential pharmacological target. TRPC3 channels can apparently be activated through different ways: in response to receptor stimulation of Gq-protein coupled receptors, such as the muscarinic acetylcholine receptors M1, M3, and M5, or through store depletion of intracellular Ca2+-stores. The aim of the present study was to characterize the impact of different TRPC3-activators on the channel's permeation properties. Therefore, we compared the permeation properties of TRPC3 when activated by two different stimuli: the cholinergic agonist Carbachol and the novel TRPC3-agonist GSK1702934A. Besides investigations with wild-type TRPC3 channels, we also intended to characterize permeation and gating as well a linkage between these functions in TRPC3G652A, a mutant of TRPC3 in which the amino acid glycine in position 652, in the transmembrane domain 6, the putative pore region of the channel, is exchanged with alanine. Whole-cell voltage clamp recordings were made from HEK293 cells, transfected stably with wild-type TRPC3 or the mutant channel TRPC3G652A. The present study shows that the exchange of G652 with alanine results in a decreased lipid-sensitivity of the channel, making G652 a decisive point for the lipid-sensitivity and therefore lipid-mediated gating of TRPC3. Furthermore, our results show that GSK is able to induce currents through wildtype TRPC3 as well as through the G652A mutant. GSK-induced currents were characterized by a change in the ion selectivity as compared to Carbachol-induced currents with both TRPC3 wildtype and TRPC3 G652A, leading us to speculate that GSK is able to influence the ion selectivity of TRPC3 channels through a “pore dilation”-process.

Abstract

Zusammenfassung:

TRPC3 ist ein Vertreter der TRP-Kanalfamilie, mit hoher Leitfähigkeit für Ca2+ und Na+. TRPC3-Kanäle können anscheinend durch mehrere Wege aktiviert werden: Entweder durch die Stimulation von Gq-gekoppelten Rezeptoren, wie zum Beispiel der muskarinischen Acetylcholin-Rezeptoren M1, M3, und M5, oder durch die Entleerung intrazellulärer Ca2+-Speicher. Das Ziel der vorliegenden Studie war es, die Einflüsse unterschiedlicher TRPC3-Aktivatoren auf die Permeationseigenschaften des TRPC3- Kanals zu untersuchen. Dazu verglichen wir die Permeationseigenschaften von TRPC3 nachdem er durch zwei verschiedene Stimuli aktiviert worden war: durch den Muskarin-Rezeptor-Agonisten Carbachol und durch den neuen TRPC3-Agonisten GSK1702934A. Außerdem wollten wir die Permeations- und Steuerungs-

Eigenschaften einer TRPC3 Mutante untersuchen: TRPC3G652A ist eine Punktmutation des TRPC3-Kanals in der die Aminosäure Glycin in Postion 652 durch Alanin ausgetauscht wurde. G652 befindet sich in der transmebranären Domäne 6, der vermuteten Porenregion des Kanals, und scheint für dessen Steuerung von Bedeutung zu sein. Alle Messungen wurden im Whole-cell voltage clamp-Modus an HEK293 Zellen durchgeführt, welche vorher mit TRPC3 oder der Mutante transfeziert worden waren. Die vorliegenden Ergebnisse zeigen dass der Austausch von G652 gegen Alanin in einer verminderten Lipidsensitivität des Kanals resultiert. G652 scheint daher ein entscheidendes Strukturelement für die Lipidsensitivität und somit auch für die Lipid-vermittelte Steuerung des TRPC3-Kanals zu sein. Weiters zeigen unsere Ergebnisse dass GSK in der Lage ist Ströme sowohl durch TRPC3 als auch durch die Mutante zu induzieren. Verglichen zu den Carbachol-induzierten Strömen jedoch zeigten GSK-induzierte Ströme eine charakteristische Veränderung der Ionenselektivität, was uns dazu veranlasst über eine GSK-induzierte „Poren- Dilatation“, also einen offenen Kanalzustand von TRPC3 mit spezifisch veränderter Ionenpermeabilität zu spekulieren.

Abbreviations

7) Abbreviations

ASIC Acid-sensing ion channel AT1 receptor Angiotensin II receptor type 1 BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid BDNF Brain-derived neurotrophic factor Cch Carbachol CHF chronic heart failure CIRB calmodulin-inositoltrisphosphate receptor binding region DAG diacylglycerol DNA Deoxyribonucleic acid DRG neurons Dorsal root ganglion neurons ER endoplasmatic reticulum GSK GSK1702934A HEK cells Human Embryonic Kidney 293 Cells IP3 inositol 1,4,5-trisphosphate IPAH Idiopathic pulmonary arterial hypertension M1 / M3 / M5 receptor muscarinic acetylcholine receptor 1 / 3 / 5 mGluR1 metabotropic glutamate receptor 1 NCX1 sodium-calcium exchanger member 1 non-SOCE non-store-operated Ca2+-Entry OAG 1-oleoyl-2-acetyl-sn-glycerol ORAI1 Calcium release-activated calcium channel protein 1 P2X receptor P2X purinoreceptor PASMC pulmonary artery smooth muscle cell

PIP2 phosphatidylinositol 4,5-bis-phosphate PI-PLC Phosphoinositide phospholipase C PKC Protein kinase C Rpm revolutions per minute SEM standard error of the mean SOCE Store operated Calcium Entry STIM1/STIM2 Stromal interaction molecule 1/2 TM transmembrane segment TrkB receptor Tropomyosin receptor kinase B VGC Voltage gated calcium channel

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