Facilitation of transmitter release from rat sympathetic neurons via presynaptic

P2Y1 receptors

Doctoral thesis at the Medical University of Vienna for obtaining the academic degree

Doctor of Philosophy

GIRI KUMAR CHANDAKA

supervisor

Prof. Dr. Stefan Boehm Department of Neurophysiology and -pharmacology Center for Physiology and Pharmacology MedicalUniversity of Vienna Waehringerstrasse 13a A-1090 Vienna Austria.

Compiled and submitted: August 2011, Vienna, Austria.

1 OM NAMAH SHIVAY

dedicated to my parents, brother and especially to my beloved wife

2 INDEX 1 ABSTRACTS………………………………………………………………………….4 1.1 ABSTRACT……………………………………………………………………...... 4 1.2 KURZFASSUNG………………………………………………………………...... 5

2 INTRODUCTION...... 6

2.1 Chemical neurotransmission...... 6 2.1.1 Neurotransmitter release...... 7 2.1.2 Neurotransmitter receptors...... 9 2.1.3 G protein-coupled receptors………………………………..……...... …11 2.1.4 Ligand-gated ion channels………………………………..…………………...... 15 2.1.4.1 Cys-loop receptor family...... …………………………..………....……..16 2.1.4.2 Glutamate receptor family...………………………………..……………...... 20

2.2 Voltage-gated ion channels...... 23 2.2.1 The voltage-gated sodium channels…………………………………...... …....24 2.2.2 The M-type K+ Channels…………………………………………………...….... 26 2.2.3 The N-type or CaV2.2 Channels……………………………………………..…...30

2.3 Nucleotides as transmitters ……………………………………………………….....35 2.3.1 P2X receptors ………………………………………………………...... 35 2.3.2 P2Y receptors ……………………………………………………………….…..36 2.3.3 E-NTPDases ………………………………………………………………….....43

2.4 Sympathetic nervous system ………………………………………….…………....45

2.5 P2 receptors in sympathetic nervous system……………………………………..…..47

3 AIMS…………………………………………………………….……………..…….50

4 MATERIALS & METHODS………………………………………………...….…...51

5 RESULTS…………………………………………………….………………...……57

6 DISCUSSION …………………….………………………….………………....…...74

7 CONCLUSIONS……………………………………………………...... ……....81

8 REFERENCES …………………………………………………..….…………...…..82

9 ABBREVIATIONS………………………………………………………………....105

10 ACKNOWLEDGEMENTS…………………………………………...... 107

11 CURRICULUM VITAE……………………………………………………..….…..108

3 1.1 ABSTRACT

P2Y1, 2, 4, 12, and 13 receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors has been missing. The search for such receptors was the purpose of this study. In primary cultures of rat superior cervical ganglion neurons and in PC12 cell cultures, currents were recorded via the perforated patch-clamp technique, and the release of [3H]noradrenaline was determined. ADP, 2-methylthio-ATP, and ATP enhanced stimulation-evoked [3H] overflow from superior cervical ganglion neurons treated with pertussis toxin to prevent the signalling of inhibitory G

proteins. This effect was abolished by P2Y1 antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca2+ stores. ADP and a

specific P2Y1 agonist caused an inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, [3H] overflow was also

enhanced by a specific P2Y1 agonist and by ADP, but only when P2Y12 receptors were blocked. ADP also enhanced K+-evoked [3H] overflow from PC12 cells treated with pertussis

toxin, but only in a clone expressing recombinant P2Y1 receptors. In addition to P2Y receptors, ectoenzymes and ion channels are also known to be expressed in the rat sympathetic neurons and it remained unclear to what extent they can associate with each other to form signalling

units. Thus P2Y1, P2Y12, P2X2 receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins and studied by FRET microscopy which showed that the G protein-coupled receptors are able to form both homo- and heterooligomers with each other.

Thus these results demonstrate that the P2Y1 and P2Y12 receptors form homo and heterooligomers at the membrane and mediate opposite effects at the rat sympathetic nerve

terminals upon agonist stimulation, P2Y1 receptors mediate a facilitation of noradrenaline release most probably by an inhibition of Kv7 channels, whereas earlier results showed that

P2Y12 receptors mediate an autoinhibition of noradrenaline release through an inhibition of voltage-gated calcium channels.

4 1.2 KURZFASSUNG

Es wurde bereits gezeigt, dass P2Y1, 2, 4, 12 und 13 Nukleotidrezeptoren präsynaptische Inhibition vermitteln, allerdings fehlt der Beleg für eine stimulierende Wirkung präsynaptischer P2Y Rezeptoren. Die Suche nach solchen Rezeptoren war das Ziel dieser Arbeit. Dafür wurden Ströme unter Verwendung der perforated patch-clamp Methode, sowie die Freisetzung von [3H]Noradrenalin in Primärkulturen von sympathischen Neuronen aus dem Ganglion cervicale superius (SCG) sowie in PC12 Zellen gemessen. ADP, 2-methylthio-ATP und ATP steigerten die stimulationsbedingte Freisetzung von [3H] in SCG Neuronen ,welche zuvor mit Pertussis Toxin behandelt worden waren, um die Aktivierung inhibitorischer G-Proteine zu unterbinden.

Diese Effekte wurden sowohl durch P2Y1 Antagonisten als auch durch Hemmung von Phospholipase C, aber nicht durch Inhibition von Proteinkinase C oder Entleerung 2+ intrazellulärer Ca Speicher verhindert. ADP und ein spezifischer P2Y1 Agonist verursachten eine Hemmung von Kv7 Kanälen. Dieser Effekt konnten durch einen entsprechenden Rezeptorantagonisten aufgehoben werden. In Zellen, die zuvor nicht mit Pertussis Toxin behandelt worden waren, steigerte ADP ebenfalls die Freisetzung von [3H], allerdings nur + nach vorheriger Blockade von P2Y12 Rezeptoren. ADP steigerte auch die durch K ausgelöste 3 [ H] Freisetzung aus PC12 Zellen, allerdings nur in einem Klon welcher hetrolog P2Y1 Rezeptoren exprimierte. Man weiß, dass zusätzlich zu den P2Y Rezeptoren in sympathischen Neuronen der Ratte auch Ektoenzyme und Ionenkanäle exprimiert werden, allerdings blieb es bis dato unklar in welchem Maße diese assoziieren und Signaleinheiten bilden können. Daher

wurden P2Y1, P2Y12, P2X2 Rezeptoren und NTPDase1 und -2 in einer mit einem fluoreszierenden Protein markierten Form hetrolog exprimiert und mit Hilfe von FRET Mikroskopie analysiert. Es zeigte sich, dass die G-Protein gekoppelten Rezeptoren in der Lage sind, sowohl Homo- als auch Hetrooligomere zu bilden. Daher zeigen diese Resultate, dass

P2Y1 und P2Y12 Rezeptoren Homo- und Hetrooligomere an der Membran bilden und bei Stimulation durch Agonisten gegenläufige Effekte and den synaptischen Endigungen von

sympathischen Neuronen der Ratte verursachen. P2Y1 Rezeptoren mediieren dabei eine Steigerung der Noradrenalin Freisetzung, höchst wahrscheinlich durch eine Hemmung von

Kv7 Kanälen, wohingegen, wie bereits frühere Ergebnisse zeigten, P2Y12 Rezeptoren eine Autoinhibition der Noradrenalinfreisetzung durch eine Hemmung von Kalzium Kanälen vermitteln.

5 2 INTRODUCTION

2.1 Chemical Neurotransmission Cells, especially neurons, communicate with each other, similarly to organisms, and the transfer of information from one neuron to another occurs at specialized sites of contact called synapses. The first neuron is said to be presynaptic and the target cell is called as postsynaptic. The process of information at the synapse is termed as synaptic transmission and is of two different types: electrical or chemical. Electrical synapses are comparatively simple in structure and function and allow the direct transfer of ionic current from one cell to the next by a special channel called gap junctions formed between two cells (Maeda & Tsukihara, 2011). Most gap junctions allow ionic current to pass in both directions, thus electrical synapses are bidirectional. Each gap junction consists of clusters of several thousands of intercellular channels referred to as gap junction channels, each of which is formed by docking of two hemichannels known as connexons. And each connexon is formed by six connexin subunits surrounding the central pore (Maeda & Tsukihara, 2011). In primitive organisms, cells communicate through chemical messengers that are secreted into the extracellular space and bind to the receptors in other cells. Similarly in higher animals like rodents, humans, etc. neurons also use a similar yet a complex and sophisticated form of chemical signaling, which is faster and precise. Neurons secrete neurotransmitters at the sites of functional contact of two neurons called chemical synapses. The presynaptic and postsynaptic membranes at chemical synapses are separated by a synaptic cleft that is approximately 20 nm wide (Schmid & Hollmann, 2010). The cleft is filled with a matrix of fibrous extracellular protein, which makes the pre and postsynaptic membranes to adhere to each other. The presynaptic side of the synapse is usually an axon terminal, which contains dozens of small membrane enclosed spheres, called synaptic vesicles, of about 40 nm size (Haucke et al., 2011). These vesicles store approximately 1500-2000 neurotransmitter molecules, which is used to communicate with the postsynaptic neuron (Haucke et al., 2011). Electrical signals in the presynaptic cell cause the release of neurotransmitter that subsequently binds to surface receptors and triggers an electrical signal, the synaptic potential, in the postsynaptic cell. The synaptic potentials in turn may evoke an action

6 potential which is conveyed along the axon to the nerve terminal to trigger the release of neurotransmitter (Wang et al., 2009). Thus, neurons communicate with each other by an alternating chain of electrical and chemical signals.

2.1.1 Neurotransmitter release In the chemical synaptic transmission, most neurotransmitters fall into 3 categories: amino acids, amines and peptides.

Amino Acids Amines Peptides

Gamma-aminobutyric Acetylcholine Cholecystokinin acid (GABA) (Ach) (CCN) Glutamate (Glu) Dopamine Dynorphin (DA) Glycine (Gly) Epinephrine Enkephalins (Enk) Histamine Neuropeptide Y

Noradrenaline Somatostatin (NA) Serotonin (5- Substance P HT) Thyrotropin- releasing hormone Vasoactive intestinal polypeptide (VIP)

Table 1 The three chemical categories of the major neurotransmitters.

7 The amino acid and amine neurotransmitters are small organic molecules and are stored in and released from synaptic vesicles, whereas peptide neurotransmitters are large molecules stored in and released from secretory granules. Neurotransmitters are released from the presynaptic nerve terminal as multimolecular packets called quanta. Each quantum is packed in a synaptic vesicle and is released into the synaptic cleft by a process called exocytosis at specialized release sites called the active zones within the presynaptic terminal (Haucke et al., 2011). Neurotransmitter release is triggered by the arrival of an action potential in the axon terminal. The depolarization of the terminal membrane causes voltage-gated calcium channels (esp. N and P/Q type) in active zones to open, thus a large inward driving force of Ca2+ is generated and Ca2+ will flood the cytoplasm of the axon terminal as long as the Ca2+ channels are open. The resulting elevation in internal calcium causes neurotransmitter release from synaptic vesicles concentrated at the release sites within active zones (Haucke et al., 2011). Exocytosis is fast because Ca2+ enters at the active zones, where synaptic vesicles are docked to release their contents. Thus, the intracellular calcium plays a major role in neurotransmitter release (Neher & Sakaba, 2008). Active zones are regions of the nerve terminal where voltage-gated calcium channels, synaptic vesicle membrane proteins and other proteins that regulate transmitter release which are collectively referred to as the cytoplasmic matrix of the active zone (CAZ), interact with each other (Haucke et al., 2011). There are several CAZ components such as Rab6 interacting protein (ELKS), liprin, GIT family proteins, Rab3 interacting molecules (RIMs), the SNARE regulator Munc13, the giant proteins bassoon and piccolo and the mammalian relatives of the Drosophila melanogaster 2+ protein bruchpilot (BRP). In the presence of high [Ca ]i these proteins alter their conformation, so that the lipid bilayer of the vesicle and presynaptic membranes fuse to form a pore that allows the neurotransmitters to escape into the cleft (Haucke et al., 2011). Vesicle exocytosis is the major and complex step through which neurotransmitters are released from vesicles, by fusing the membranes of the vesicles and presynaptic terminal, as described above. This complex process of specific binding and fusion of membranes is carried out by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, that were first identified in yeast cells. There are several members of SNAREs such as; 25 members in Sacchromyces cerevisae, 36 members in Homo sapiens and 54 members in Arabidopsis thaliana (Jahn & Scheller, 2006; Malsam et al.,

8 2008). The majority of these proteins face the cytoplasm, followed by a membrane spanning domain and the rest of it faces the lumen of the intracellular compartment or the extracellular surface. Depending on their function, SNAREs have been classified into two different types operating on opposite membranes: on a transport vesicle (v) and target (t) membrane SNAREs, (Malsam et al., 2008). Vesicle priming is dependent on ATP, calcium ions that act on synaptotagmin that tightens the binding, leading to vesicle fusion and transmitter release.

Voltage activated calcium channels of the CaV2 family are the functional link between action potentials and exocytosis (Zamponi, 2003). The released transmitter is rapidly removed, to avoid an accumulation in the synaptic cleft, which could cause a prolonged stimulation and thus a pronounced desensitization of the postsynaptic receptors. Thus, neurotransmission is terminated by three different mechanisms: a) receptor desensitization in the continued presence of the transmitter, b) transmitters like acetylcholine and ATP are degraded by specific enzymes (acetylcholine esterase and NTPDase family of enzymes) in the synaptic cleft (Snyder, 2006; Abbracchio et al., 2009) and c) transmitters like norepinephrine, serotonin and amino acid neurotransmitters such as GABA, glutamate and glycine are taken up either by the nerve terminal they have been released from or into surrounding glia cells by transporters (Snyder, 2006). Some of these are the targets for pharmacological intervention.

2.1.2 Neurotransmitter receptors Neurotransmitters released from the presynaptic terminal into the synaptic cleft affect the postsynaptic neuron by binding to the specific receptors present on it, causing a conformational change in the protein that might alter the protein function. The neurotransmitter receptors can be grouped into two different types: ionotropic and metabotropic receptors. Ionotropic receptors change the membrane conductance for specific ions and thereby depolarize or hyperpolarize the membrane. One of the reasons for the generation of an action potential is the depolarization of the membrane, whereas hyperpolarization may inhibit the propagation of action potentials by lowering the membrane potential away from firing threshold.

9 Instantaneous response of ionotropic receptors to the released neurotransmitters are transient (milliseconds), thus this process is termed as neurotransmission, whereas activation of metabotropic receptors, leads to long lasting effects (seconds to minutes) through complex signaling mechanisms. Their effects range from changing the gating properties of ion channels to changes in the expression levels of proteins, thus this slow process is termed as neuromodulation (Schicker et al., 2008). Ionotropic receptors are also termed as ligand-gated ion channels that are specialized for rapidly converting extracellular chemical signal back into electrical signal at chemical synapses. These are membrane-spanning proteins consisting of three to five subunits that form a pore (Schicker et al., 2008). In the absence of a neurotransmitter, these pores are usually closed, but when neurotransmitter binds to specific sites on the extracellular region of the channel, it induces a conformational change which leads to the opening of pore. The functional consequence of this depends on which ions can pass through the pore. Like any other channel, ionotropic receptors are also selective for certain ions and as mentioned earlier, this selectivity determines the nature of the postsynaptic response. Neurotransmitters like acetylcholine, glutamate and serotonin stimulate cation channels, causing an influx of sodium and calcium ions that depolarizes the postsynaptic cell from resting membrane potential, which brings the membrane potential towards the threshold for generating action potentials, this effect is known as excitatory. A transient postsynaptic membrane depolarization caused by the presynaptic release of neurotransmitter is called an excitatory postsynaptic potential (EPSP) and thus these transmitters that are responsible are referred as excitatory neurotransmitters. Whereas, transmitters like γ-aminobutyric acid (GABA) and glycine act on channels that are permeable to chloride ions that leads to hyperpolarization of the postsynaptic cell from resting membrane potential, thus bringing the membrane potential away from threshold for generating action potentials, hence this effect is known as inhibitory. A transient hyperpolarization of the postsynaptic membrane potential caused by the presynaptic release of transmitter is called an inhibitory postsynaptic potential (IPSP) and thus such transmitters are referred to as inhibitory neurotransmitters (Hille, 2001).

10 2.1.3 G protein-coupled receptors The superfamily of G-protein-coupled receptors (GPCRs) is one of the largest families of proteins in the mammalian genome, which are very diverse in structure and function. Approximately half of the drugs available in the market are targeted at these receptors. There are a wide variety of ligands for these receptors which include ions, amines, peptides, proteins, lipids, nucleotides, organic odorants and photons (Fredriksson et al., 2003). The two main characteristics for a protein to be classified as a GPCR are: the ability of the receptor to interact with a G-protein and presence of seven sequence stretches of about 25 to 35 consecutive residues which represent seven α-helices spanning the plasma membrane. Thus these receptors are also referred to as seven transmembrane (TM) receptors. More than 800 GPCR sequences have been identified so far. They have been further divided into five main families: glutamate (G), rhodopsin (R), adhesion (A), frizzled/taste2 (F) and secretin (S), thus known as the GRAFS classification system. In addition, there are also 23 protein sequences known that are not grouped in either of the above mentioned families. Each of the five families is again divided further into groups (Fredriksson et al., 2003; Schioth & Fredriksson, 2005).

The secretin receptor family consists of 15 members and the name is related to the fact that the secretin receptor was the first one to be cloned in this family. The N-terminus is between ~60 and 80 amino acids long, important for ligand binding. The members of this family include calcitonin receptor (CALCR), the corticotropin-releasing hormone receptors (CRHRs), the glucagon receptor (GCGR), the gastric inhibitory polypeptide receptor (GIPR), the glucagon-like peptide receptors (GLPRs), the growth hormone-releasing hormone receptor (GHRHR), pituitary adenylyl cyclase-activating protein (PACAP), the parathyroid hormone receptors (PTHR), the secretin receptor (SCTR), and the vasoactive intestinal peptide receptor (VIPR) (Fredriksson et al., 2003; Schioth & Fredriksson, 2005).

The adhesion receptor family is the second largest and consists of 33 members. The family is also known as EGF-TM7, representing the epidermal growth factor (EGF) domains in the N-terminus.

11 The name adhesion receptor family relates to the long N-terminus of variable lengths from 200 to 2800 amino acids, which contains motifs that mediate cell adhesion. Members include the brain specific angiogenesis-inhibitory receptors (BAIs 1-3), GPR56, GPR64 (HE6), EMR (1-4), CD97, ETL, the lectomedin receptors (LECs 1-3), GPR (123-125), CELSR (1-3), GPR (110, 111, 113, 115, 116, 133 and 144) (Fredriksson et al., 2003; Schioth & Fredriksson, 2005).

The glutamate receptor family consists of 15 members, which include 8 metabotropic

glutamate receptors, two GABA receptors (GABABR1 and GABABR2), a calcium-sensing receptor (CASR) and five taste receptors type 1 (TAS1). The ligand binding N-terminus is approximately 280 to 580 amino acids in length (Fredriksson et al., 2003; Schioth & Fredriksson, 2005).

The frizzled/taste2 receptor family has 24 members which are divided into two distinct clusters, the frizzled and the TAS2 receptors and there is a very little similarity between the two. There are 13 TAS2 receptors found in human and mouse databases, which are known to be expressed in the tongue and palate epithelium and are known to mediate the function as bitter taste receptors. They have a very short N-terminus which is unlikely to contain a ligand binding domain. The frizzled family of receptors consists of 10 frizzled receptors (FZD 1-10) in addition to smoothened (SMOH). The N-terminus of frizzled receptors is approximately 200 amino acids long and might have a role in Wnt binding and they control cell fate, proliferation and polarity during metazoan development by mediating signals from secreted glycoproteins (Wnt) (Fredriksson et al., 2003; Schioth & Fredriksson, 2005).

The rhodopsin family (R) is the largest family with several hundred members. The structure is different from other receptors discussed until now, as they have a short N-terminus. The ligand binding site of rhodopsin family members, with a few exceptions is in a cavity between the transmembrane domains, unlike other GPCRs where N-termini plays major role. The rhodopsin family of receptors is divided into four main groups: α, β, γ and δ (Fredriksson et al., 2003; Schioth & Fredriksson, 2005).

12 The α-group of rhodopsin receptors consists of 89 members and is divided into five main branches: the prostaglandin receptor cluster (15), amine receptor cluster (40), opsin receptors cluster (9), melatonin receptor cluster (3) and melanocortin, endothelial differentiation sphingolipid, cannabinoid and adenosine (MECA) receptor cluster (22). The β-group of rhodopsin receptors consists of 36 members with no further divisions. All the known ligands to these receptors are peptides. The γ-group of rhodopsin receptors consists of three main branches: the suboesophageal ganglion (SOG) receptor cluster (15), melanin-concentrating hormone (MCH) receptor cluster (2), and the chemokine receptors cluster (42). The δ-group of rhodopsin receptors is the largest among the rhodopsin family with an estimated 518 members and is divided into four branches: MAS-related receptor cluster (8), glycoprotein receptor cluster (8), purine receptor cluster (42), and the olfactory receptor cluster (460) (Fredriksson et al., 2003; Schioth & Fredriksson, 2005). The purine receptor cluster consists of the formyl peptide receptors (FPRs), a large number of orphan GPCRs and the nucleotide P2Y receptors (Fredriksson et al., 2003). In the present study, two different P2Y receptors have been investigated and a detailed description of P2Y receptors is given in chapter 2.3.2.

Signaling via GPCRs Unlike ionotropic receptors that directly convert a chemical to an electrical signal, metabotropic receptors mode of action is indirect via heterotrimeric G-proteins that can interact with various enzymes or ion channels (Schicker et al., 2008). All the metabotropic receptors can mediate slow, long lasting and diverse postsynaptic actions via G-protein coupled receptors (GPCRs), which involves three steps, neurotransmitter molecules bind to the receptors in the postsynaptic membrane, the receptor proteins activate small proteins called G-proteins present in the intracellular space of the postsynaptic membrane, activate effector proteins. GPCRs are also referred to as metabotropic receptors, because they can trigger widespread metabolic effects. One of the most important features of GPCRs is that, though different chemical signaling molecules bind to them, yet all the GPCRs share a common molecular structure. GPCRs have an extracellular glycosylated N-terminal, seven membrane spanning hydrophobic domains (thus,

13 these receptors are also known as seven-transmembrane receptors) and an intracellular C- terminus and all the G-proteins have the same basic mode of operation. The C-terminal domain and the intracellular loops interact with the G-proteins and it seems as if the third loop was the major determinant for G-protein selectivity (Wess, 1998). G-proteins are heterotrimers and have three subunits, α, β and γ. In the resting state, a guanosine

dipohosphate (GDP) molecule is bound to the Gα subunit and when this GDP bound G- protein binds to a certain receptor that already has a transmitter molecule bound to it, then the G-protein exchanges GDP for GTP from the cytosol. The activated GTP-bound G-protein

splits into two: the Gα subunit plus GTP and the Gβγ complex. Both of them are capable of interacting with downstream effector proteins and initiating signals (McCudden et al., 2005).

Gα subunit is an enzyme that breaks down GTP to GDP, thus terminating its own activity by

converting the bound GTP to GDP, thus the Gα and the Gβγ subunits come back together,

available for a new cycle. G-proteins are generally referred to by the nature of their Gα

subunits. Thus, GS contains αS subunit, Gi contains αi subunit and Gq contains αq subunit. In

addition, another simple function that is known to divide G-proteins is; GS designating that

the G-protein is stimulatory and Gi designating that the G-protein is inhibitory. Till date, there are 16 α, 5 β and 12 γ subunits known and all the known trimers operate via the same general mechanism (Pierce et al., 2002; McCudden et al., 2005). The most important effects mediated by G-proteins are: regulation of adenylyl cyclase activity (Harden et al., 2010), activation of phospholipase C (PLC) (Bunney & Katan, 2011), modulation of voltage gated potassium channels and voltage gated calcium channels (VGCC’s) (Filippov et al., 2010).

Adenylyl cyclase can be stimulated by GS proteins and inhibited by Gi proteins (Harden et al., 2010), it is a membrane bound enzyme that synthesizes the second messenger cyclic AMP (cAMP) from ATP and cAMP mainly exerts its effects by activating protein kinase A (PKA), though there are other pathways for its mode of action (Skroblin et al., 2010).

Inositol 1,4,5-triphosphate (IP3) is another important second messenger, which is generated

by the membrane bound enzyme phospholipase C (PLC) that is mainly activated by Gq

proteins. PLC hydrolyses membrane bound phosphotidylinositol 4,5-bisphosphate (PIP2) to 2+ produce IP3 and diacylglycerol (DAG). IP3 causes the release of Ca from the endoplasmic

reticulum by activating endoplasmic IP3 receptors. DAG can be cleaved further by a

membrane bound enzyme, phospholipase A2 (PLA2) to release arachidonic acid that can act

14 as a messenger and can also mediate the activation protein kinase C (PKC). PKC, in turn is able to phosphorylate a number of target proteins, such as ion channels, thus regulating their function (Delmas et al., 2002; Suh & Hille, 2002; Ford et al., 2003; Zhang et al., 2003; Ford et al., 2004; Gamper et al., 2004).

2.1.4 ligand-gated ion channels As described above, ligand-gated ion channels participate in fast synaptic transmission,

which include nicotinic acetylcholine, 5-hydroxytryptamine3 (5-HT3), γ-aminobutyric acidA

(GABAA), glycine, ionotropic glutamate and P2X receptor families. These ligand-gated ion channels are further divided into 3 families: Cys-loop receptor family, glutamate receptor family and the P2X receptor family (Collingridge et al., 2009). A detailed discussion about the first two receptor families is given below, whereas the P2X receptors are described in detail in chapter 2.3.1.

Figure 1 Schematic representation of the three families of ligand-gated ion channel subunits.

15 The pentameric Cys-loop receptor superfamily includes nicotinic acetylcholine receptors

(nAChRs), 5-hydroxytryptamine3 (5-HT3) and zinc-activated (ZAC) receptors are cation

selective ion channels and the γ-aminobutyric acidA (GABAA) and glycine (Gly) receptors are anion selective channels. The tetrameric glutamate receptor family consists of N-methyl- D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. The third category represents the trimeric ATP-gated P2X family of receptors. The large coloured cylinders represent the transmembrane domains. Yellow circles indicate the cysteine residues participating in disulphide bond formation and red cylinders indicate α-helical regions involved in ion conductivity/selection. Figure taken from (Collingridge et al., 2009).

2.1.4.1 Cys-loop receptor family Cys-loop receptors are membrane-spanning ion channels activated by neurotransmitters, which are responsible for fast excitatory and inhibitory neurotransmission in the peripheral

and central nervous systems. Nicotinic acetylcholine, 5-HT3, GABAA, Glycine and zinc- activated (ZAC) receptors are grouped in this receptor family. Cys-loop receptors are the major targets for treatment of neurological disorders such as Alzheimer’s, anxiety, epilepsy, learning, attention deficit and drug addiction. They are also the major targets for many active compounds like anaesthetics, muscle relaxants and insecticides. All the members of this receptor family share a common structure and the name of this family is derived from a 13- amino acid loop within the extracellular domain (ECD) that is closely enclosed by a pair of disulphide-bonded Cys residues (Betz & Laube, 2006; Thompson et al., 2010). As shown in figure 1, they consist of five pseudo-symmetrically arranged subunits (pentamer) surrounding a central ion-conducting pore. Each subunit has a large extracellular N-terminal domain, the transmembrane domain (TMD) consists of four membrane spanning α-helices (M1-M4), an intracellular domain (ICD) primarily formed by the large M3-M4 intracellular loop (~ 100- 270 residues) and a relatively small extracellular C-terminal domain (Betz & Laube, 2006; Olsen & Sieghart, 2008; Barnes et al., 2009; Collingridge et al., 2009; Olsen & Sieghart, 2009; Changeux, 2010; Thompson et al., 2010). The ECD contains the ligand-binding sites and thus is a major pharmacological target. The TMD enables ions to cross through the

16 membranes and is the target for compounds such as alcohols, anaesthetics and steroids. The ICD is responsible for receptor modulation, sorting and trafficking and contains the opening that allows ions access in and out of the pore and also influences the ion conductance (Thompson et al., 2010).

Nicotinic acetylcholine receptors (nAChR) Acetylcholine (ACh) is one of the most important neurotransmitters in the central (CNS) and peripheral nervous (PNS) systems and it is produced by the enzyme choline acetyltransferase. ACh acts on two different receptors: the G protein coupled muscarinic acetylcholine receptors (mAChRs) and the nicotinic acetylcholine receptors (nAChRs). nAChRs are ligand-gated ion channels that are present both in CNS and PNS (Taly et al., 2009; Changeux, 2010). nAChRs are transmembrane oligomers with a molecular mass of ~290 kDa, consisting of five homo or heteromeric subunits symmetrically arranged around a central ion channel, encoded by 17 genes. Out of the 17, nine α-subunits and three β-subunits are expressed in the brain. The various pentameric subunit compositions differ in their localization and also in their pharmacological and kinetic properties. There are 2-5 ACh or nicotine binding sites within the extracellular domain, which depends on the pentameric composition and are distinct from the functionally linked cationic ion channel. Thus the existence of diverse range of neuronal nAChR hetero-oligomers creates substantial challenges for targeted drug design (Taly et al., 2009; Changeux, 2010). Activation of brain nAChRs results in the enhanced release of different neurotransmitters such as dopamine, serotonin, glutamate and GABA. The effect of ACh results in the fast opening (micro to millisecond range) of a cationic channel that is permeable to Na+, K+ and sometimes Ca2+. Chronic exposure of ACh or nicotinic drugs leads to a gradual decrease in the rate of this ionic response (100 ms to minutes), which leads to a high-affinity, densensitized, closed state of the receptor. Thus, many of the clinical drugs targeting nAChRs are administered for months, resulting in long-term changes in receptor properties and/or number. Chronic exposure to nicotine results in an approximately two fold increase in the total number of high-affinity receptors. Thus there is a growing interest in modulating

17 nAChRs to treat various nervous system disorders such as alzheimer’s disease, schizophrenia, depression, attention deficit hyperactivity disorder and tobacco addiction (Taly et al., 2009; Changeux, 2010).

5-hydroxytryptamine3 (5-HT3) receptors 5-hydroxytryptamine (5-HT; serotonin) is one of the major neurotransmitters and there are 7

different classes of 5-HT receptors identified (5-HT1 to 5-HT7). Most of the 5-HT receptors

are metabotropic that couple to G-proteins, except for the 5-HT3 receptors which are ionotropic (Engleman et al., 2008).

The 5-hydroxytryptamine type-3 (5-HT3) receptor is a cation selective channel that mediates neuronal depolarization and excitation within the central and peripheral nervous systems. It was originally described as the M-type receptor due to its effects on mesenteric or gut

contraction. 5-HT3 belongs to the Cys-loop receptor family which is assembled as a pentamer of subunits which surround a central ion channel (Barnes et al., 2009; Collingridge et al., 2009). M2 lines the intramembraneous portion of the ion conduction pathway (cations esp. Na+ and K+), surrounded by M1, M3 and M4 that separates it from membrane lipid, in addition amino acids within an α-helical portion of the large intracellular loop between M3- M4 also contributes to the ion permeation pathway, but is not required for receptor function.

The 5 known subunits of 5-HT3 receptors are 5-HT3A, 5-HT3B, 5-HT3C, 5-HT3D and 5- HT3E. The 5-HT3A and 5-HT3B receptor subunit share a 41% amino acid sequence identity. Whereas, 5-HT3C and 5-HT3E share a 36% and 39% amino acid identity with the human 5- HT3A subunit. 5-HT3C, 5-HT3D and 5-HT3E subunits require 5-HT3A for their trafficking to the cell membrane or forming a ligand binding site (Barnes et al., 2009; Collingridge et al., 2009).

The 5-HT3 receptor is found throughout the central nervous system and in higher concentrations in brain stem areas like area postrema, nucleus tactus solitarius (NTS) and dorsal motor nucleus of the vagus nerve that are important for the initiation and coordination

of the vomiting reflex. Thus antagonism of 5-HT3 receptors at this site would prevent chemotherapy-induced post-operative nausea and vomiting. It was also shown that

presynaptic 5-HT3 receptors facilitate the release of glutamate onto dorsal vagal preganglionic, nucleus tractus solitatius and area postrema neurons (Barnes et al., 2009). 5-

18 HT3 receptors are known to regulate dopaminergic (DA) neurotransmission in many brain areas including mesolimbic, mesocortical and nigrostriatal DA pathways. Recent evidence

indicates that ethanol can directly bind to the 5-HT3 ion channel and enhance their ion

current flow and it was also shown that direct activation of 5-HT3 receptors probably is one mechanism by which ethanol and other drugs of abuse increases the release of DA in the

mesolimbic system. Thus, 5-HT3 receptors are an important site for pharmacological treatment of alcohol and drug abuse (Engleman et al., 2008).

GABAA receptors Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain,

which mediates its effect via ionotropic GABAA receptors (GABAA-R) and metabotropic

GABAB receptors (GABAB-R). GABAB receptors are sensitive to selective agonist baclofen,

in addition to GABA, but are not sensitive to GABAA receptor ligands such as bicuculline

and muscimol. GABAA-Rs were first identified pharmacologically as being activated by GABA and the selective agonist muscimol. They are blocked by by bicuculline and picrotoxin and are modulated by benzodiazepines, barbiturates and other CNS depressants.

GABAA-Rs are found throughout the mammalian nervous system and play a role in almost all the brain physiological functions, thus serve as target for numerous classes of drugs

(Olsen & Sieghart, 2008; 2009). GABAA receptors are pentameric ligand gated ion channels grouped under Cys-loop family of receptors. Thus similar to other Cys-loop family members,

GABAA-Rs are also formed by five subunits surrounding a central pore that forms the ion

channel. The GABAA-Rs are one of the most complex ion channels, due to the existence of

19 genes for GABAA receptors: α1-6, β1-3, γ1-3, δ, ε, θ, π and 3 ρ subunits that are

sometimes called GABAC receptors. These 19 subunits assemble as heteropentamers that result in a complex subtype heterogeneity in structure, which determines their

pharmacological profile. It was shown that most of pentameric GABAA receptors are formed from two copies of a single α subunit, two copies of a single β subunit and one copy of either of the γ, δ or ε subunits. Though there is an estimate that more than 800 subtypes might exist in the brain, but only 11 subtypes have been conclusively identified. Each subtype can play a significant role in the cells in which they are expressed (Olsen & Sieghart, 2008; 2009).

19 Glycine receptors (GlyR) Glycine is one of the major neurotransmitters at the inhibitory synapses, where it activates strychnine-sensitive glycine receptors (GlyRs). Thus strychnine, a convulsive alkaloid from the Indian tree Stychnos nux vomica antagonizes the binding of glycine to GlyR. Strychnine is mostly used to distinguish glycinergic inhibition from GABAergic. The GlyRs are the first neurotransmitter receptor proteins to be isolated from mammalian CNS (Betz & Laube, 2006). GlyRs respond to agonist stimulation by increasing the chloride conductance of the postsynaptic cell. There are only four known vertebrate isoforms of the GlyR α-subunit (α1-

α4) and a single β-subunit, with a probable stoichiometry of α13β2 or α14β. GlyRs are prominently expressed in medulla oblongata, pons and spinal cord and they also have a role to play in retina and inflammatory pain sensitization (Betz & Laube, 2006; Thompson et al., 2010). GlyR α-subunit gene expression is developmentally and regionally regulated. GlyR α1 mRNA and protein are specifically expressed in spinal cord, brainstem and colliculi of adult rodents, whereas α2 transcripts are present in abundance in hippocampus, cerebral cortex and thalamus at birth, but decrease in an adult. Appreciable levels of α3 transcripts are found in spinal cord, cerebellum and olfactory bulb. The Glrb gene is widely expressed in CNS both during the embryonic and postnatal periods (Betz & Laube, 2006).

Zinc-activated (ZAC) receptors The fifth and not so popular receptors, the genes of which are found only in human and dog, but not in at least some rodents are the zinc-activated (ZAC) receptors, that are grouped under Cys-loop family of receptors. This protein contains all the motifs that are characteristic of Cys-loop ligand gated ion channel subunits and has 411 amino acid residues. Transcripts of ZAC subunits were found in human placenta, trachea, spinal cord, stomach and fetal brain (Davies et al., 2003; Collingridge et al., 2009).

2.1.4.2 Glutamate receptor family Glutamate (Glu) is one of the major excitatory neurotransmitters, and glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Glutamate receptors are localized on both the neuronal and non-neuronal cells. The mammalian

20 glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels. Glutamate receptors are tetramers of more than >900 residues, which form a central ion pore and all the known members of this family share a similar architecture as shown in figure 1. Each subunit contains four domains: the extracellular amino-terminal domain, the extracellular ligand-binding domain (LBD), the transmembrane domain (TMD) and an intracellular carboxy-terminal domain. The functional tetrameric glutamate receptors are formed exclusively by the assembly of subunits within the same functional receptor class. Glutamate receptors are divided into four different classes based on pharmacology and structural homology: AMPA, kainate, NMDA and δ receptors (Traynelis et al., 2010).

AMPA receptors AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPA-Rs) mediate the majority of fast excitatory synaptic transmission in the brain. There are four genes that are known to encode glutamate receptors and are represented as GluA1-4 and the tetrameric ligand-gated ion channel is formed by the proteins encoded by these four genes. Most of the native AMPA-Rs are heterotetramers made of at least two of the four subunits, GluA1-4. However, when coexpressed an individual AMPA-R subunit can also assemble into a functional homotetrameric ion channel. Auxiliary subunits such as stargazin/TARPs (transmembrane AMPA-R regulatory proteins), cornichon 2/3 and CKAMP44 (cysteine-knot AMPA-R modulating protein) (also known as Shisa9) are also known to attach to the receptor core and each auxiliary subunit has a distinct function on receptor gating and/or trafficking. Thus the complexity of AMPA-Rs in brain is further increased by their co- assembly with the auxiliary subunits. The function of AMPA-Rs is critical for synaptic plasticity and their dysfunction leads to several neurological and psychological diseases such as X-linked mental retardation, Alzheimer’s disease, amyotrophic lateral sclerosis, limbic encephalitis, ischemic brain injury and Rasmussen’s encephalitis (Collingridge et al., 2009; Nakagawa, 2010).

21 Kainate receptors (KARs) Kainate receptors (KARs) are also activated by glutamate and are highly expressed in central nervous system. There are five subtypes known: GluK1, GluK2, GluK3, GluK4 and GluK5, which co-assemble in different combinations to form functional receptors and the subunit names to the corresponding genes, are: GRIK1, GRIK2, GRIK3, GRIK4 and GRIK5. The progress in understanding KARs has lagged behind compared to the other glutamate receptors due to the lack of specific pharmacological tools. The other major problem is that the KAR agonists including kainate (a glutamate analogue isolated from the seaweed Digenea simplex), acts on both KARs and AMPARs and there are no specific KAR specific antagonists available and if available not yet in clinical use. Several functional studies indicate that KARs mainly have a modulatory role in synaptic transmission rather than being the major postsynaptic target for the synaptically released glutamate similar to AMPARs and NMDARs. Several studies with antagonists against GluK1 suggest that the subunit has an important role to play in many neurological disorders such as in epilepsy, neurodegenration, pain, migraine and also in psychiatric conditions (Collingridge et al., 2009; Jane et al., 2009).

NMDA receptors (NMDARs) One of the tetrameric glutamate receptor subtypes that also mediates fast excitatory neurotransmission and sensitive to N-methyl-D-aspartate (NMDA) are the NMDA receptors (NMDARs). The functional subunits of rats and humans have been cloned and there are three families of subunits identified: GluN1, GluN2 and GluN3. GluN2 family consists of four members GluN2A-D subunits and GluN3 includes two subunits GluN3A and GluN3B. The large NMDAR protein complex has multiple binding sites for different ligands, an NMDA binding site, a strychnine-insensitive glycine binding site and a binding site within the channel for non-competitive antagonists. The binding sites for polyamines, protons, redox reagents, zinc and magnesium present on the receptor complex can modulate its function. NMDARs are expressed majorily in the cortical and hippocampal regions and are very important in learning and memory. NMDARs play a major role in many memory tasks, including those using spatial, reference, working and passive avoidance memory and also in long-term potentiation (LTP), a cellular phenomenon involved in some types of memory. Compared to other glutamate receptors, NMDARs are said to be the most vulnerable to the

22 ageing process and these changes are suggested to have an impact on learning and memory abilities (Collingridge et al., 2009; Magnusson et al., 2010; Traynelis et al., 2010).

δ receptors There are two members of this family, Gluδ1 and Gluδ2 (Collingridge et al., 2009; Traynelis et al., 2010). Though they are grouped under glutamate or glutamate like receptor family because of the high amino acid sequence identity, they are not known to bind glutamate or get activated by glutamate to produce a current. The endogenous ligands still remain unknown, which were classified for many years as orphans (MacLean, 2009; Swanson & Sakai, 2009). However, there are reports suggesting the role of Gluδ2 receptors in controlling synapse formation and synaptic plasticity in purkinje fibers of the adult mice (Yuzaki, 2004). Gluδ2 knock out studies revealed that, there is impairment in synaptic plasticity, stabilization, elimination, motor control and learning. Thus Gluδ2 receptor plays an important role in cerebellar function (Hirano, 2006).

2.2 Voltage gated ion channels The ion transport mechanisms might have developed during evolution simultaneously during the process of the membrane formation, which allowed the vital ions to pass through the membrane in either direction (Hille, 2001). Although the pore theory existed from as early as 1840s that was proposed by various biophysicists like Von Brücke, Helmholtz, Ludwig, DuBois Reymond Fick and others to explain osmosis, but the term channel or ion channel was finally coined by Bertil Hille in 1967 (Hille et al., 1999). Much before the channels were identified, Sidney Ringer in 1880’s suggested that sodium, potassium and calcium are the most important ions and their presence in a definite concentration is essential for the longevity of a frog heart in vitro or for that matter these ions play an important role in the excitability of nerve and muscle cells. During the last century, the major cellular roles for each of the ions in ringer’s solution were discovered and ever since the list of vital ions has accelerated. In the nervous system, the excitation and electrical signaling involves the movement of Na+, K+, Ca2+ and Cl- ions through their respective channels and the response of the channel to stimulus is called the gating, which is nothing but a simple opening or closing of the pore within the channel. The pore has the most important function of selective

23 permeability and allows only the specific ions to pass down their electrochemical activity gradients at a very high rate of more than 106 ions per second (when considered from a molecular point of view) (Hille, 2001). One of the most famous experiments in electrophysiology was that on the squid giant axon, by Hodgkin and Huxley in 1952, where they recognized three different components of the current called sodium, potassium, leakage and later calcium; the channel names such as Na+, K+ and Ca2+ channels were given later which are widely accepted now. Ever since their discovery, the list of ion channels had been continually growing and there are more than 100 identified genes for Na, K and Ca channels only, in a mammal like rat, thanks to molecular genetics. 10 years later, during 1960s the voltage-clamp technique was developed elsewhere, which revealed two major permeability mechanisms distinguished by their ion selectivities and clear separable kinetics, which are Na+ selective and K+ selective that showed voltage dependent kinetics. They were also the first two ion channels that were recognized and + described in detail (Hille, 2001). In the present study, M-type (KV7) K channels have been studied.

2.2.1 The Voltage gated Sodium channels (NaV) The sodium channels are members of the superfamily of ion channels, which includes potassium and calcium channels. Unlike its other above mentioned superfamily members, the various known sodium channels do not differ in their functional properties. After a lot of confusion finally, a standardized nomenclature has been proposed based on the nomenclature of voltage-gated potassium channels that is widely accepted (Catterall et al., 2005). This nomenclature uses a numerical system to define subfamilies and subtypes based on the amino acid sequence similarities between the channels. According to this nomenclature, name of the channel consists of the chemical symbol of the principal permeating ion (Na), with the

principal physiological regulator (voltage), indicated as a subscript (NaV) (Catterall et al.,

2005). The gene subfamily has been indicated as a number following the subscript (NaV1), which is the only known subfamily. The number following the full point indicates the

specific channel isoform (eg: NaV1.1) and this last number has been assigned based on the order in which each gene was identified (Catterall et al., 2005).

24

Figure 2 Sodium channel subunits. The cylinders represent the α-helical segments. Bold lines represent polypeptide chains of each subunit. The extracellular domains β1 and β2 subunits are shown as immunoglobuluin-like folds. Ψ, depict sites of probable N-linked glycosylation. P in the circles represents the phosphorylation sites of protein kinase A and in the diamonds for protein kinase C. Figure taken from (Catterall et al., 2005).

NaV channels are formed by one α-subunit of approximately 260 kDa. The α-subunits contain four homologous domains (I-IV) similar to voltage gated calcium channels and each domain consists of six α-helical transmembrane segments (S1-S6), in addition to a membrane- reentrant loop between the S5 and S6 segments which forms the lining of the pore (Lai & Jan, 2006; Catterall, 2010). The S4 segment serves as the voltage sensor (Lai & Jan, 2006; Catterall, 2010) and the short intracellular loop between domains III and IV forms the channel inactivation gate (Catterall et al., 2006). The α-subunits are associated with β- subunits (β1-β4) (Lai & Jan, 2006), which are of 33-36 kDa (Catterall et al., 2006; Catterall, 2010). Each β-subunit consists of a single transmembrane segment and an extracellular domain (Lai & Jan, 2006). Although the pore forming α-subunits are sufficient for functional expression, the kinetic, and voltage dependence of channel gating depend on auxiliary β-

subunits (Catterall, 2010). Ten genes encode the α-subunits in mammals; NaV1.1 to NaV1.9,

25 in addition to an atypical sodium channel referred to as Nax, which has more than 50%

identity with other NaV proteins (Lai & Jan, 2006). Although there are exceptions, but

NaV1.1, NaV1.2, NaV1.3 and NaV1.6 are mainly expressed in the central nervous system,

NaV1.4 and NaV1.5 are found in the cardiac and skeletal muscle systems and NaV1.7, NaV1.8

and NaV1.9 are found in the peripheral nervous system (Lai & Jan, 2006). NaV channels are highly concentrated at the axon initial segment (AIS) in axons. In axons, Nav channels

principally, NaV1.2 and NaV1.6 are responsible for action potential initiation at the AIS and nodes of ranvier. They are also responsible for action potential propagation along the unmyelinated axon and action potential back propagation in dendrites (Lai & Jan, 2006).

2.2.2 The M-type K+ channels There are several families of K+ channel pore forming α-subunit genes that have been cloned and identified; and each of these families contain a large number of members (Robbins, 2001). The International Union of Pharmacology (IUPHAR) has proposed a new nomenclature in 2003 (Gutman et al., 2003), which is being followed ever since. Among the several different families known, K+ channel subunits with 6TMDs and a single P-loop are the largest, the earliest discovered and the best characterized (Robbins, 2001). This family 2+ + includes the KV channels, in addition to the Ca -activated K channels (KCa). All the members’ share common structural characteristics such as, both the N and C termini of these proteins are intracellular and there is a P region between the fifth and sixth transmembrane domains for the ionic conductance. A functional channel is formed by the tetrameric (homo- or hetero-meric) association of these 6TM/1P subunits, within the family members (Gutman et al., 2003).

26 Figure 3 Structure of the 6TMD/1P family of K+ channels. The numbered rods represent the transmembrane domains (TMDs). P-loop is between 5th and 6th TMDs. N and C indicates the N- and C-termini of the protein that are intracellular. Figure adapted from (Delmas & Brown, 2005).

+ Inward rectifying K (Kir) and the KATP channels are grouped together under a second class of K+ channels, which have 2TM domains and a single P region in-between these two TM’s. The functional channel is formed by a tetrameric association of these 2TM/1P subunits. Similar to the first class of K+ channels, their N- and C-termini are also intracellular (Gutman et al., 2003).

Large conductance channel, Slo or Slow Poke, has been grouped under the third class which has 7TM domains, which has a P region between the sixth and seventh TM’s and like the previous classes of channels, these channels also function as a tetramer. C-terminus is located intracellular, but N-terminus is extracellular (Gutman et al., 2003). The fourth class of proteins has a 6TM/1P segment linked in tandem to a 2TM/1P segment and the functional channel is formed by the dimeric association of this 8TM/2P subunit (Gutman et al., 2003).

The K2P family is another class which contains two 2TM/1P containing subunits linked in tandem and the functional channel is a dimeric association of 4TM/2P subunits (Gutman et al., 2003). The Kv7 channels studied in the present project belong to the 6TM/1P family of voltage- + gated K channels, described above. The M-current (IM) was first described by David Brown in bull frogs (Brown & Adams, 1980) and in rat sympathetic neurons (Constanti & Brown, 1981). They were one of the first currents, which were known to be inhibited by the neurotransmitter acetylcholine that acts on muscarinic acetylcholine receptor (mAChR), thus

the currents were named after the receptors as, M-currents (IM) and the ion channel as M- channel respectively (Brown & Adams, 1980; Delmas & Brown, 2005). 20 years after their discovery, the molecular composition of M-channels was put forward, which belonged to the + subunits of the KV7/KCNQ family of K channels (Wang et al., 1998). Out of the five known

members of this family KV7.1 to KV7.5, KV7.2 and KV7.3 subunits are known to form the

27 components of the native M-channel in most neurons (Wang et al., 1998) and sometimes

homomeric KV7.2 subunits were also found to form the M channel (Hadley et al., 2003;

Schwarz et al., 2006), although with a suspected contribution of KV7.5 subunit (Shah et al.,

2002). KV7.2 and KV7.3 subunits are probably assembled as a two plus two heterotetramer

(Cooper et al., 2000; Jentsch, 2000; Hadley et al., 2003; Delmas & Brown, 2005). KV7.3 and

KV7.5 subunits form heteromultimers in several sensory neurons in dorsal root ganglia that

are known to express KV7.2, KV7.3 and KV7.5 subunits (Passmore et al., 2003; Brown &

Passmore, 2009). However, it is believed that all KV7 subunits including the cardiac KV7.1 channels, when associated as homomeric channels, can form M channels, defined kinetically and pharmacologically (Delmas & Brown, 2005). Thus, all the homomeric channels, similar to their heteromeric counter parts show a similar time and voltage-dependent gating (Selyanko et al., 2000) and are inhibited by the M-channel blockers like linopirdine and by the activation of GPCRs, such as M1 muscarinic acetylcholine (Selyanko et al., 2000;

Shapiro et al., 2000; Delmas & Brown, 2005) and P2Y6 receptors (Boehm, 1998; 2003b).

Among the five KV7 family members, except for KV7.1 which are expressed in the heart,

peripheral, epithelial and smooth muscle cells, the rest of the four KV7 channels are known to be expressed exclusively in the nervous system (Brown & Passmore, 2009); with an

exception of KV7.4 subunits that are also predominantly found in auditory and vestibular systems (Hansen et al., 2008). The M-channel has a key role in regulating the excitability of various central and peripheral neurons including sympathetic, hippocampal pyramidal and striatal neurons, by acting as a brake on repetitive action potential discharges. In various neurons, activation of M-channels during an initial stage of action potential discharge serves to suppress the later action potentials by sustained depolarization (Brown & Passmore, 2009). Thus, there is a strong enhancement of repetitive firing, when the M-channels are blocked by drugs like linopirdine or XE991. Whereas, the enhancement of M-current with drugs like retigabine hyperpolarizes the neuron and inhibits spike generation (Lechner et al., 2003; Brown & Passmore, 2009). Kv7 channels have been linked to Long QT syndrome, deafness and imprinting (leads to

developmental abnormalities) (Robbins, 2001), but mutations in the genes for KV7.2 and

KV7.3 in particular have been linked to epilepsy. Benign familial neonatal convulsions

(BFNC), a form of juvenile epilepsy is caused as a result of mutations in the genes for KV7.2

28 or KV7.3 (Jentsch, 2000). BFNC is a rare disease with an incidence of 1 in 100,000 people and the seizures usually disappear within a few weeks or months after birth. It is believed that a total of 20-30% of the currents is reduced in BFNC, which is critical during the postnatal development that results in seizures that might probably be compensated by other K+ channels in later life. In either case, the BFNC mutations do not seem to alter either ion selectivity or gating (Jentsch, 2000).

Pathways modulating M-channels

Like several other ion channels, KV7 channels also require a certain level of

phosphatidylinositol-4,5-bisphosphate (PIP2) in the cell membrane to open. Initially, Suh and

Hille showed that wortmannin that inhibits phosphoinositide 4-kinase (PI4K) (a PIP2 synthesizing enzyme), also inhibits and slows the recovery of M-currents in sympathetic

neurons and recombinant KV7.2/7.3 channels from the inhibition by muscarinic acetylcholine

receptors (Suh & Hille, 2002). Later another group also showed that PIP2 is required for the M-channel activity (Zhang et al., 2003) Since the discovery of M-channels, it always seemed likely that the closure of the channels is an indirect mechanism mediated by different receptors which use common intermediate

secondary messengers. Further research in this direction revealed that Gq (α-subunit in

particular) and/or G11-linked GPCRs upon activation could close M-channels (Pfaffinger, 1988; Brown et al., 1989; Lopez & Adams, 1989; Caulfield et al., 1994; Haley et al., 1998; Haley et al., 2000). The two most popular pathways for the closure of M-channels by the

stimulation of Gq–coupled GPCRs are via the activation of muscarinic and bradykinin receptors, in either case, the principle effect of stimulating these GPCRs is the activation of

phospholipase-Cβ (PLCβ) that leads to the hydrolysis of membrane bound PIP2 to produce 2+ inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 mediates the release of Ca ions from intracellular stores and DAG activates protein kinase C (PKC) (Delmas & Brown, 2005). Among the two said pathways for the closure of M-channels, channel-inhibition by the activation of muscarinic acetylcholine receptors (mAChR) is well studied and is known since the channel’s discovery. After a lot of confusion regarding the mode of action of mAChR’s, finally it is now believed that M-channel inhibition via the stimulation of mAChR’s is due to

29 the depletion of membrane PIP2 because of its hydrolysis rather than because of its accumulated by products. On the other hand, bradykinin receptors which are closely

associated with IP3 receptors (IP3R), unlike mAChR’s, upon stimulation, mediate the release of Ca2+ ions from the intracellular stores (Cruzblanca et al., 1998; Delmas & Brown, 2002; 2+ Delmas et al., 2002). The released Ca ions bind to KV7 attached calmodulin (CaM) and

close KV7 channels. One can argue, similar to the mAChR pathway, why don’t bradykinin receptors also follow the same pathway, by closing the M-channels, immediately after 2+ membrane-PIP2 depletion. The reason could be that the release of intracellular Ca might

stimulate PIP2 synthesis, which might prevent PIP2 depletion, through the activation of the neuronal calcium sensor (NCS1) protein that binds Ca2+ and activates PI4 kinase (PI4K) (Zhao et al., 2001; Burgoyne et al., 2004). The reason for mAChR not acting in a similar way

is that, unlike bradykinin receptors, mAChR’s are not closely associated with IP3R’s, thus do 2+ not release sufficient intracellular Ca ions to initiate PIP2 synthesis (Delmas & Brown, 2005). Thus M-channels are controlled in several ways such as by several receptors, neurotransmitters and hormones. There are several G protein coupled P2Y receptors that are known to modulate currents

through KV7 channels (Lechner & Boehm, 2004): P2Y1 (Filippov et al., 2006), P2Y2

(Filippov et al., 1994), P2Y4 (Meng et al., 2003) and P2Y6 (Boehm, 1998; Bofill-Cardona et al., 2000) receptors. It was a debate until recently; about the mechanism of P2Y receptors in

inhibiting KV7 channels. But recently it was shown that the KV7 channel inhibition by P2Y receptors is by Ca2+/CaM action, similar to bradykinin pathway (Bofill-Cardona et al., 2000; Zaika et al., 2007).

2.2.3 The N-type or CaV2.2 channels Calcium channels are one of the most important and well studied voltage gated ion channels. Voltage-gated calcium channels together with voltage-gated potassium and sodium channels are members of a gene superfamily of transmembrane ion channel proteins. Upon membrane depolarization, voltage-gated calcium channels mediate calcium influx and regulate several processes like secretion, neurotransmission, contraction and gene expression. Biochemical characterization of calcium channels revealed that they are complex proteins composed of

four or five distinct subunits such as α1, α2, β, γ and δ that are encoded by multiple genes.

30 Among the 5 different subunits, α1 subunit is the largest one with 190-250 kDa and incorporates the conduction pore, voltage sensor and gating apparatus. It also consists of the known sites for channel regulation by second messengers, drugs and toxins (Catterall et al.,

2003; Catterall & Few, 2008). The α1 subunit is composed of about 2000 amino acid residues arranged in four homologous domains: I to IV. Each domain consists of 6 transmembrane α helices: S1-S6 and a membrane associated P loop between S5 and S6. The segments S1 through S4 serve as voltage sensor module, whereas the transmembrane segments S5 and S6 in each domain and the P loop in between them form the pore (Catterall & Few, 2008). There

are four known auxiliary protein subunits, with which an α1 subunit can be associated to form a functional channel. The intracellular hydrophilic β subunit is of 50-65 kDa. The

transmembrane, disulfide-linked α2δ complex, encoded by a single gene results in a prepolypeptide, which is posttranslationally cleaved and disulfide-bonded to yield the mature

α2 and δ subunits (Catterall & Few, 2008). The β subunit and the α2δ subunit complex are components of most types of Ca2+ channels (Catterall et al., 2003). In addition, a γ subunit is also found in skeletal muscles and the related subunits are expressed in heart and brain. Although the auxiliary subunits have important function in the Ca2+ channels such as trafficking and on several biophysical properties (Dolphin, 2003), the pharmacological and

electrophysiological diversity of the channels arises from the existence of multiple α1

subunits (Catterall & Few, 2008). Mammalian α1 subunits are encoded by at least 10 different genes. After a lot of debate, a rational nomenclature was put forward based on the well defined K+ channel nomenclature. Calcium channels were named using the chemical symbol of the permeating ion (Ca), with the principle physiological regulator (voltage) as

CaV. Thus, 10 different genes encoding the α1 subunits are further divided into 3 subfamilies: 2+ CaV1, CaV2 and CaV3. Based on their biophysical characteristics, voltage-gated Ca channels are further classified into two different types: high-voltage-activated (HVA) and low-voltage-activated (LVA) channels. As the names indicate, HVA channels require larger

membrane depolarizations to get activated, whereas LVA require lower. Both CaV1 and CaV2

channel families have been grouped under HVA, and LVA consists of CaV3 channels

(Zamponi et al., 2010). A complete study of amino acid sequences of α1 subunits revealed a more than 70% identity within a family but less than 40% when compared between families (Catterall et al., 2003).

31

Figure 4 Subunits of the CaV channels. Helices are represented as cylinders. The lengths of lines approximately correspond to the lengths of polypeptide segments. The voltage-sensing module is depicted in yellow and the pore forming module shown in green. Figure taken from (Catterall & Few, 2008).

2+ CaV1 subfamily consisting of members CaV1.1 to 1.4, mediates L-type Ca currents. CaV1.1 is found to be expressed in skeletal muscle transverse tubules and mediates cellular functions

such as excitation-contraction coupling. CaV1.2 is found in cardiac myocytes, endocrine

cells, neuronal cell bodies and proximal dendrites and CaV1.3 are found in endocrine cells,

neuronal cell bodies and dendrites. CaV1.2 and 1.3 share common cellular functions that include excitation-contraction coupling, hormone release, regulation of transcription and

synaptic integration. The last member of this family CaV1.4 is found in retina and its

functions include neurotransmitter release from rods and bipolar cells. CaV1 subfamily of channels is the molecular targets of the organic calcium channel blockers that are widely used in cardiovascular disease therapy. All the members of this family share common specific antagonists, such as dihydropyridines, phenylalkylamines and benzothiazepines (Catterall et al., 2003). Phenylalkylamines are intracellular channel blockers, which are thought to block the channel from the cytoplasmic side. Their receptor site is formed by

32 amino acid residues in the S6 segments in domains III and IV. Dihydropyridines are both channel activators and inhibitors, therefore are thought to shift the channel towards open or closed state. Their receptor site includes amino acid residues in the S5 segment of domain III and S6 segment of domains III and IV. Benzothiazepines bind to a third receptor site, which is thought to overlap with those of phenylalkylamine binding (Catterall et al., 2003).

CaV2 subfamily consists of 3 members: CaV2.1 to 2.3, which mediate P/Q-, N- and R-type 2+ Ca currents. CaV2.1 and CaV2.2 channels are found in nerve terminals and dendrites and also share common cellular functions such as neurotransmitter release and dendritic Ca2+

transients. CaV2.3 channels are found in neuronal cell bodies and dendrites and mediate

repetitive firing. Compared to other families, CaV2 family members have very specific

antagonists. CaV2.1 is specifically blocked by ω-agatoxin IVA from funnel web spider

venom. The CaV2.2 channels are blocked by ω-conotoxin GVIA and related cone snail

toxins. Whereas, CaV2.3 channels are specifically blocked by the toxin SNX-482 derived from tarantula venom (Catterall et al., 2003).

2+ CaV3 subfamily includes 3 members: CaV3.1 to 3.3, which mediate T-type Ca currents. All

the CaV3 subfamily members share a common tissue distribution such as, neuronal cell

bodies, dendrites and cardiac myocytes, with an exception of CaV3.3 channels that are not reported in cardiac myocytes. They are also reported to share common functions like

pacemaking and repetitive firing. CaV3 subfamily members are comparatively insensitive to

all the antagonists listed for CaV1 and CaV2 subfamily members. The peptide kurtoxin is

reported to inhibit the activation of CaV3.1 and CaV3.2 channels (Catterall et al., 2003).

2+ CaV2.2 channels have been studied in the present project, which mediate N-type Ca currents. These are most important at synapses formed by neurons of the peripheral nervous 2+ system. It is actually the Ca entering through CaV2.2 channels which is primarily responsible for initiating synaptic transmission at fast synapses (Dunlap et al., 1995; Catterall & Few, 2008). The roles of Ca2+ ions, Ca2+ channels and the most important proteins that mediate neurotransmitter release have already been described in detail in an earlier chapter

(2.1.1 Neurotransmitter release). CaV2.2 channels are extensively regulated by protein

33 2+ kinases, Gβγ subunits and SNARE proteins. G proteins are known to control various Ca

channels, including CaV2.2 channels. Neurotransmitters released from presynaptic nerve

terminals bind to GPCRs and mediate an inhibition of CaV2.2 channels, which results in the reduction of neurotransmitter release (Catterall & Few, 2008). Many of the neurotransmitters

such as acetylcholine, adenosine, noradrenaline, glutamate, GABA, amines, prostaglandin E2, somatostatin and neuropeptides, inhibit Ca2+ channels in a similar way (Boehm, 2003a). The 2+ pertussis toxin sensitive Gi/Go class of G-proteins usually mediates an inhibition of Ca

currents, upon the release of their Gβγ subunits. Particularly, the activation of Gi-coupled

P2Y12 receptors are known to mediate an inhibition of CaV2.2 channels that leads to an autoinhibition in the noradrenaline release from the sympathetic neurons (Lechner et al., 2+ 2004). Such type of Ca channel regulation is caused by the binding of Gβγ subunits directly to the Ca2+ channel (Herlitze et al., 1996; Ikeda & Dunlap, 1999; Catterall & Few, 2008). 2+ Several studies have suggested three sites for interaction of Gβγ subunits with Ca channel α1 subunits: the N-terminus (Canti et al., 1999), the intracellular loop connecting domains I and II (Herlitze et al., 1997; Zamponi et al., 1997) and the C-terminus (Qin et al., 1997; Furukawa et al., 1998; Li et al., 2004). In addition to the above mentioned, voltage-

dependent inhibition of CaV2 channels by direct interaction with Gβγ subunits, many neurons also exhibit a voltage-independent approach, which depends on intracellular signaling pathways (Catterall & Few, 2008). This type of voltage-independent Ca2+ channel inhibition is usually mediated by Gq coupled G-proteins, by activating PLCβ which hydrolyzes

membrane PIP2 (Delmas et al., 2005). Earlier studies also showed that synaptic protein

interaction (synprint) site and Gβγ subunits bind to a distinct portion of syntaxin-1A and the

presence of syntaxin-1A is a prerequisite to inhibit CaV2.2 channels via G proteins (Catterall & Few, 2008). There are several studies suggesting the roles of nucleotides in regulating the functions of

CaV2.2 channels by stimulating G protein coupled P2Y receptors (Lechner & Boehm, 2004):

P2Y1 (Gerevich et al., 2004), P2Y2 (Abe et al., 2003), P2Y12 (Vartian & Boehm, 2001) and

P2Y13 (Wirkner et al., 2004) receptors. Among the above mentioned P2Y receptors, P2Y1

and P2Y2 receptors are Gq coupled thus, their mode of action is by activating PLCβ which

depletes membrane PIP2, as described previously. Whereas, P2Y12 and P2Y13 receptors are

34 Gi coupled, which upon activation release their Gβγ subunits that probably directly bind to the Ca2+ channels and inhibit them, as described earlier.

2.3 Nucleotides as transmitters ATP was established as a transmitter after a lot of controversies and the term “purinergic” was put forward by Geoffrey Burnstock in 1972 (Burnstock, 1972; 2007). ATP is degraded to ADP and adenosine by ectonucleotidases (Abbracchio et al., 2009) and these breakdown products activate different purinergic receptors, which were first defined in 1976 (Burnstock, 1976; 2007). Further, two types of purinoceptors were identified, P1 receptors selective for adenosine and P2 receptors activated by adenine and uridine nucleotides and nucleotide

sugars (Burnstock, 2007). There are four different subtypes of P1 receptors, A1, A2A, A2B and

A3, which are members of rhodopsin like family of G-protein coupled receptors. In transmembrane domains (TMI-TMVII) human adenosine receptors share 11-18% sequence

identity with P2Y receptors. P1 receptors couple to adenylate cyclase. A1 and A3 are

negatively coupled to adenylate cyclase through the Gi/o, whereas A2A and A2B are positively

coupled to adenylate cyclase through Gs. The human A2B receptor is also known to couple

through Gq/11 to regulate phospholipase C and A3 receptor may interact with Gs directly. P1 and P2Y receptors are often expressed in the same cells (Burnstock, 2007).

2.3.1 P2X receptors P2 receptors are activated by purines and some subtypes of pyrimidines. At least 15 different P2 receptors are known to be expressed in mammals that regulate a broad range of physiological responses (Burnstock, 2007; Harden et al., 2010). However, two different families of cell surface receptors mediate the actions of extracellular nucleotides, P2X and P2Y. P2X receptors are ligand-gated ion channels that conduct extracellular cations and thus contribute to synaptic transmission (Robertson et al., 2001), when activated by ATP (Khakh

& North, 2006). P2X receptors consist of seven different subunits, P2X1-7. Each subunit is thought to consist of intracellular N- and C-termini that possess binding motifs for protein kinases; two transmembrane domains (TM1 and TM2), TM1 is involved in channel gating

35 and TM2 lining the ion pore; a large extracellular loop, with 10 conserved cysteine residues forming a series of disulfide bridges; a hydrophobic H5 region close to the pore vestibule, for possible receptor/channel modulation by ions and an ATP binding site, which may involve regions of the extracellular loop close to TMI and TM2. This topology is the simplest among ionotropic receptors and is also the simplest stoichiometry among ionotropic receptors. All the 7 different subtypes of P2X receptors show a 30-50% sequence identity at the peptide level (North, 2002; Stojilkovic et al., 2005; Egan et al., 2006; Roberts et al., 2006; Jarvis & Khakh, 2009; Browne et al., 2010). All the P2X receptors are trimers and activated by three molecules of ATP. Only a small portion of the ion channel is submerged within the lipid bilayer and a bulk of the receptor protein is extracellular (~280 a.a.) (Browne et al., 2010). These subunits form channels as homotrimer or as heterotrimers (North, 2002). P2X receptors mediate a wide range physiological functions that range from thrombosis through gantrointestinal motility, afferent sensation (chemoreception and taste), renal autoregulation, neuropathic pain, bone resorption and inflammation (Browne et al., 2010).

2.3.2 P2Y receptors P2Y receptors are a group of eight molecularly defined G-protein coupled receptors (GPCR) with 7 transmembrane (TM) domains (Abbracchio et al., 2006). P2Y receptors consist of 308 to 377 amino acids with a molecular weight of 41 to 53 kDa after glycosylation. Unlike P2X receptors, P2Y receptors are activated not only by ATP, but also by ADP, UDP and UTP. The first P2Y receptors were cloned in 1993 (Lustig et al., 1993; Webb et al., 1993). Since then, several P2Y subtypes have been isolated by homology cloning and assigned a subscript on the basis of cloning chronology and accordingly there are eight different P2Y receptors till

date: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14 (Abbracchio et al., 2003; Abbracchio et al., 2006; Burnstock, 2007). The missing numbers in the P2Y receptor choronology represent either non-mammalian orthologs or receptors having some sequence homology to P2Y receptors, but no functional evidence available (Burnstock, 2007).

The P2Y1, P2Y2, P2Y4 and P2Y6 receptors activate Gq/phospholipace C-β and share 35-52%

primary sequence homology, thus can be grouped into a P2Y1 receptor subfamily (Harden et

al., 2010). P2Y11 receptor is the only subtype which is selectively activated by ATP, similar

to P2X receptors and is linked to Gq and Gs and is also considered a member of the P2Y1

36 receptor subfamily although there is only 28-30% identity to the four other members of this family (Harden et al., 2010).

The second sub group of P2Y receptors is known as P2Y12 receptor subfamily, where

P2Y12, P2Y13 and P2Y14 receptors are grouped together and are Gi/o coupled. They exhibit a sequence homology of 45-50% and have a very little sequence identity (20-25%) with the

P2Y1 receptor subfamily members (Harden et al., 2010).

Figure 5 The P2Y receptors. The members of P2Y1-like subfamily couple to Gαq G-

proteins and activate PLC-β. P2Y11 receptor couples to Gαq G-proteins and activates PLC-β

and it also couples to Gαs G-proteins, thus activates adenylyl cyclase (AC). The three

members of the P2Y12-like subfamily couples to the Gαi/o G-proteins and inhibit AC activity. Figure adapted from (Harden et al., 2010).

P2Y1 receptors

P2Y1 receptors from human, rat, mouse, cow, chick, turkey and Xenopus have been cloned and characterized (Abbracchio et al., 2006; Burnstock, 2007). ADP is more potent as an agonist than ATP, in addition to their 2-methylthio derivatives, which are even more potent, but UTP, UDP, CTP and GTP are known to be inactive (Waldo & Harden, 2004). The most potent and selective agonist known is the N-methanocarba analog of 2-MeSADP, MRS2365

(Chhatriwala et al., 2004). ATP is also known as a partial agonist at the P2Y1 receptor

37 (Palmer et al., 1998), thus at low P2Y1 receptor expression ATP will act as an antagonist (Leon et al., 1997; Hechler et al., 1998; Abbracchio et al., 2006). The antagonists MRS2179, MRS2279 and MRS2500 are more potent, compared to the previously known antagonists A3P5P and A3P5PS (Boyer et al., 1996; Boyer et al., 1998; Boyer et al., 2002). Site-directed

mutagenesis studies on the human P2Y1 receptors revealed that amino acid residues in TM 3, 6 and 7 are critical determinants in the binding of ATP and other nucleotide derivatives (Moro et al., 1998; Abbracchio et al., 2006; Burnstock, 2007). Earlier studies also showed that four cysteine residues in the extracellular loops conserved in the P2Y receptors are

essential for proper trafficking of the P2Y1 receptors to the cell surface (Hoffmann et al.,

1999). P2Y1 receptor couples to Gαq and Gα11 and the activation of this receptor increases 2+ the intracellular levels of IP3 levels via PLC-β and thus releases Ca from intracellular stores

in a PTX-insensitive manner. P2Y1 receptors are expressed in almost all the human tissues, which include, the brain, heart, placenta, lungs, liver, skeletal muscle, kidneys, pancreas, spleen, pituitary gland, lymphocytes, adipose tissue and various blood cells (Ayyanathan et al., 1996; Janssens et al., 1996; Leon et al., 1996; Moore et al., 2001). Within the brain,

P2Y1 mRNA was found to be highest in the nucleus accumbens, putamen, caudate nucleus and striatum, with lower levels expressed in the hippocampus, parahippocampal gyrus, globus pallidus, cingulated gyrus and hypothalamus (Moore et al., 2000).

As mentioned earlier, P2Y1 receptors are widely distributed in the central and peripheral nervous system and mediate a plethora of effects including the modulation of voltage- and transmitter-gated ion channels (Hussl & Boehm, 2006). In several types of neurons derived

from either the central or peripheral nervous system, P2Y1 receptors were found to control ion channels (Hussl & Boehm, 2006), to mediate presynaptic inhibition in sensory neurons (Gerevich et al., 2004; Rodrigues et al., 2005; Heinrich et al., 2008) and spinal cord neurons

(Heinrich et al., 2008). In hippocampal neurons, activation of P2Y1 receptors also leads to an

inhibition of KV7 channels (Filippov et al., 2006), and activation of these channels by e.g. retigabine reduces transmitter release from hippocampal nerve terminals (Martire et al.,

2004). Presynaptic P2Y1 receptors are also known to mediate an inhibition of glutamate release (Rodrigues et al., 2005) and to mediate platelet aggregation (Gachet, 2008).

38 P2Y2 receptors

P2Y2 receptors also known earlier as P2U have been cloned from human, rat, mouse, canine and porcine cells or tissues (Lustig et al., 1993; Parr et al., 1994; Bowler et al., 1995; Rice et al., 1995; Chen et al., 1996; Zambon et al., 2000; Shen et al., 2004). ATP and UTP act as

equipotent agonists for P2Y2 receptors, whereas ADP and UDP are not that effective

(Burnstock, 2007). Suramin acts as a weak competitive antagonist for human and rat P2Y2 receptors. AR-C126313 and related aminotetrazole derivative AR-C118925, flavanoids and tangeretin have been claimed to be more effective antagonists (Burnstock, 2007). Similar to

P2Y1 receptors, P2Y2 receptors are also Gq-coupled and activate PLCβ to produce IP3 and 2+ DAG, to release Ca from intracellular stores and activate PKC. P2Y2 receptors are known to be expressed in skeletal muscle, heart and some brain regions and at more moderate levels

in spleen, lymphocytes, macrophages, bone marrow, and lung. P2Y2 receptors have been shown to be present in very low levels in liver, stomach, pancreas, vascular smooth muscle and endothelial cells (Ralevic & Burnstock, 1998; Moore et al., 2001). In smooth muscle

cells P2Y2 receptor expression is upregulated by interleukin-1β, interferon-γ and tumour

necrosis factor-α (Hou et al., 2000), in addition P2Y2 receptor upregulation has been shown to promote nucleotide induced activation of PKC, cyclooxygenase and MAPK (Seye et al.,

2002). P2Y2 receptors have been shown to inhibit bone formation by osteoblasts (Hoebertz et al., 2002) and also inhibit N-type calcium currents in neurons (Brown et al., 2000).

P2Y4 receptors

Human, rat and mouse P2Y4 receptors have been cloned and characterized (Communi et al., 1995; Nguyen et al., 1995; Stam et al., 1996; Bogdanov et al., 1998; Webb et al., 1998; Lazarowski et al., 2001; Suarez-Huerta et al., 2001). UTP is the most potent agonist of the

recombinant human P2Y4 receptors (Nicholas et al., 1996). GTP and ITP are also known

P2Y4 receptor agonists, but 10 times less potent than UTP (Communi et al., 1996a). ATP

acts as an agonist at the rat P2Y4 receptor, but behaves as a competitive antagonist for human

P2Y4 receptor (Kennedy et al., 2000). Up4U (INS365) and dCp4U (INS37217) are agonists of

the human P2Y4 receptor. Reaction blue 2 at 100 µM effectively blocks rat P2Y4 receptors,

but partially blocks human P2Y4 receptors. Another common P2 receptor antagonist, suramin

is a weak antagonist for P2Y4 receptor (Abbracchio et al., 2006). An earlier study on

39 chimeric human/rat P2Y4 receptors showed that the structural determinants of agonism versus antagonism by ATP are located in the N-terminus and the second extracellular loop of the receptor (Herold et al., 2004). Xenopus p2y8 and turkey p2y could represent as the

orthologs of mammalian P2Y4 receptor though their sequence is distantly related. The turkey p2y receptor is coupled to the stimulation of PLC and inhibition of adenylyl cyclase,

suggesting a dual coupling to Gq/11 and Gi/o (Abbracchio et al., 2006). UTP inhibited the N- 2+ type Ca currents in the sympathetic neurons expressing P2Y4 cDNA and this inhibition was abolished by PTX (Filippov et al., 2003). It also has been shown from the same neurons that

UTP inhibited the currents through KV7 channels in a PTX-sensitive way. Human P2Y4 receptor was shown to be expressed in placenta, intestine, human unbilical vein endothelial cells, peripheral blood leukocytes, fetal cardiomyocytes and various cell line derived from

the human lung (Burnstock & Knight, 2004). High levels of P2Y4 mRNA was found in intestine, pituitary and brain, low levels were found in liver, bone marrow (Moore et al., 2001), monocyte and lymphocytes (Jin et al., 1998).

P2Y6 receptors

Human, mouse and rat P2Y6 receptors have been cloned (Chang et al., 1995; Communi et al., 1996b; Lazarowski et al., 2001). UDP is the most potent agonist for these receptors, in addition to other uridine and adenine nucleotides. The rank order of potency of various nucleotides is as follows: UDP > UTP > ADP > 2-MeSATP > ATP (Communi et al., 1996b).

Earlier experiments have shown that recombinant P2Y6 receptor couples to Gq/11 (Chang et

al., 1995; Robaye et al., 1997). Compared to other P2Y receptors, P2Y6 receptor has short C- terminal sequence, which contains a single threonine and misses the Ser333 and Ser334 that play a key role in the UTP dependent phosphorylation, desensitization and internalization of

the receptor (Robaye et al., 1997; Brinson & Harden, 2001). P2Y6 receptors are known to be expressed in placenta, spleen, thymus, intestine (Communi et al., 1996b), vascular smooth muscle, lungs (Ralevic & Burnstock, 1998), human bone, two osteoblastic cell lines, brain derived cell lines (Burnstock & Knight, 2004), kidney, adipose, heart and some brain regions

(Moore et al., 2001). P2Y6 receptor functions include regulation of chemokine production and release in monocytes (Warny et al., 2001), a role in proliferation of lung epithelial tumour cells (Schafer et al., 2003), mediate contractions of human cerebral arteries (Malmsjo

40 et al., 2003) and interaction with the tumour necrosis factor-α (TNF-α) related signals to

prevent apoptotic cell death (Kim et al., 2003). In rat sympathetic neurons, P2Y6 receptors

when activated by uridine nucleotides, inhibit currents through KV7 channels and stimulate the release of noradrenaline (Boehm, 1998; Bofill-Cardona et al., 2000).

P2Y11 receptors

P2Y11 receptor is unique among all other P2Y receptors: P2Y11 receptor gene contains an intron in the coding sequence, its natural agonist is ATP but has a very low potency and it is dually coupled to PLC and adenylyl cyclase stimulation (Abbracchio et al., 2006). It is the only P2Y receptor that does not have a rodent ortholog. The rank order of potency of various nucleotides is ARC67085 ≥ ATPγS ≈ BzATP > dATP > ADP (Communi et al., 1997; Communi et al., 1999; Qi et al., 2001). Suramin behaves as a competitive antagonist of the

human P2Y11 receptors (Communi et al., 1999). Human P2Y11 receptors when activated lead

to an increase of cAMP and IP3 via the dual activation of GS and Gq/11, which is also a unique

feature of this receptor (Communi et al., 1997; Communi et al., 1999). P2Y11 receptors are found in brain, spleen, lymphocytes, intestine and almost all other tissues (Moore et al.,

2001). Similar to other P2Y receptors, P2Y11 receptor has also a wide variety of functions at tissue level which includes role in maturation, migration of dendritic cells (Wilkin et al., 2001; Schnurr et al., 2003), granulocytic differentiation (Communi et al., 2000) and secretory role in pancreatic duct epithelial cells (Nguyen et al., 2001).

P2Y12 receptors

The human, rat and mouse P2Y12 receptors have been identified and characterized almost at the same time (Foster et al., 2001; Hollopeter et al., 2001; Savi et al., 2001; Zhang et al., 2001). ADP is the natural agonist of this receptor, whereas there are few reports suggesting ATP and its triphosphate analogs also as potent agonists. The rank order of agonist potency for diphosphates is 2-MeSADP > ADP > ADPβS. However, ATP and its analogs were found

to act as agonists either in native P2Y12 receptor expressing cells or in some heterologously expressing cells (Simon et al., 2001; Zhang et al., 2001). There are also reports suggesting

that ATP and its triphosphate analogs act as antagonists of the P2Y12 receptors in both human

and mouse platelets (Gachet, 2001; Kauffenstein et al., 2004). The P2Y12 receptor is mostly

41 expressed in the megakaryocytes and platelets, where it is the target of the antiplatelet drug

clopidogrel (Savi et al., 2001; Savi & Herbert, 2005). P2Y12 receptor plays a major role in

thrombus formation (Gachet, 2005). The P2Y1 receptor causes platelet shape change, but

aggregation occurs only when P2Y12 receptor is activated. Only coactivation of these two receptors initiates signaling pathways that ultimately trigger the activation of glycoprotein IIb/IIIa, which in turn promotes high affinity binding to fibrinogen and platelet aggregation.

P2Y12 receptor also plays a role in the recruitment of the platelets to the site of injury and in the enhancement of the efficiency of platelet activation by other agonists, such as thrombin and thromboxane A2 (Gachet, 2005). Thus this receptor is defective in some patients with blood coagulation disorders (Hollopeter et al., 2001; Cattaneo et al., 2003). Hence, numerous

selective antagonists for the P2Y12 receptors have been developed as antithrombotic drugs. Ticlopidine, clopidogrel and prasugrel are potent antithrombotic drugs of the thienopyridine family of compounds. Potent direct competitive antagonists are also known which includes the AR-C69931MX compound or also known as cangrelor that is also used in the present

study to inhibit P2Y12 receptors. Other AR-C compounds also exist, which are all ATP analogs and many are currently under clinical trails (Abbracchio et al., 2006). Ticagrelor,

another potent P2Y12 blocker, previously known as AZD6140 (AstraZeneca, Wilmington, DE) is the first oral agent in a new chemical class of nonthienopyridine antiplatelet agents termed cyclopentyltriazolo-pyrimidines (Abergel & Nikolsky, 2010). In addition to the

platelets, the P2Y12 receptors are also known to be expressed in subregions of the brain (Hollopeter et al., 2001), glial cells (Bianco et al., 2005), brain capillary endothelial cells (Simon et al., 2001), spinal cord, smooth muscle cells (Wihlborg et al., 2004) and chromaffin

cells (Ennion et al., 2004). Earlier reports also showed that P2Y12 receptors upon activation by ADP mediate an inhibition of voltage gated calcium channels and also mediate an inhibition of noradrenaline release from rat sympathetic neurons (Lechner et al., 2004).

P2Y13 receptors

The human, mouse and rat P2Y13 receptors have been identified and characterized (Communi et al., 2001; Zhang et al., 2002; Fumagalli et al., 2004). The natural agonists of this receptor

are ADP and Ap3A. ATP was also shown as a weak partial agonist for P2Y13 receptors, but

similar to P2Y1 receptors described earlier, the activity of ATP may vary according to the

42 level of expression of the P2Y13 receptors in different recombinant systems. Apart from ADP, 2-MeSADP was also shown to be a potent agonist (Abbracchio et al., 2006). The

selective P2Y12 receptor antagonist, cangrelor is also known to act as a selective P2Y13 receptor antagonist. MRS2211, a derivative of PPADS was also shown to selectively

antagonise the human P2Y13 receptors, in addition to Ap4A and 2-MeSAMP (Abbracchio et

al., 2006). The P2Y13 receptors are found in spleen, brain regions, liver, pancreas, bone marrow, heart, peripheral leucocytes (Communi et al., 2001; Zhang et al., 2002).

P2Y14 receptors

The P2Y14 receptors, previously also known as GPR105 or UDP glucose receptor and as the old name suggests, it is activated by UDP-glucose, UDP-galactose, UDP-glucuronic acid and also UDP-N-acetylglucosamine, but not by uridine or adenine nucleotides (Chambers et al.,

2000). P2Y14 receptors are 47% identical to P2Y12 and P2Y13 receptors which are grouped

together (Abbracchio et al., 2006). There are no selective antagonists available for P2Y14 receptors till now. The rat and mouse orthologs show 80 and 83% amino acid identity with

human P2Y14 receptors and also show similar agonist pharmacology (Charlton et al., 1997;

Freeman et al., 2001). Similar to other P2Y receptors, P2Y14 receptors are also expressed in wide variety of tissues or organs in human body such as placenta, adipose tissue, stomach, intestine, brain regions (corpus striatum, cerebellum, caudate nucleus, hippocampus, hypothalamus and glial cells), spleen, lung, heart, bone marrow and thymus (Chambers et al.,

2000; Moore et al., 2003). P2Y14 receptors also play a role as a chemoattractant receptor in bone marrow hematopoietic stem cells (Lee et al., 2003); in addition these receptors also play a role in neuroimmune function (Moore et al., 2003) and in dendritic cell activation (Skelton et al., 2003).

2.3.3 E-NTPDases As described above, extracellular nucleotides modulate a wide variety of tissue functions. Receptors for these nucleotide diphosphates and nucleotide triphosphates are P2X and P2Y receptors. E-NTPDases (Ecto-Nucleoside Triphosphate Diphosphohydrolases) also previously known as ecto-apyrases, NTPases or E-ATPases are a family of enzymes that

43 hydrolyze both nucleoside tri- and di-phosphates. To date eight members of this family have been described; E-NTPDases 1-8 and are present in almost every cell in the vertebrate body (Abbracchio & Burnstock, 1998). Among the eight known E-NTPDases 1, 2, 3 and 8 are expressed on the cell membrane, whereas 4, 5, 6 and 7 found in the cell interior (Robson et al., 2006; Yegutkin, 2008). E-NTPDase1 also known as apyrase earlier, is the first member of E-NTPDase family, whose molecular identity was unraveled in mide 1990s, thus was cloned and identified as a lymphocyte cell activation antigen, CD39 (Robson et al., 2006; Yegutkin, 2008). Earlier it was thought that there existed a single member in this NTPDase family that might undergo post-translational modifications, but soon a molecular relative was cloned that showed functional properties of an ecto-ATPase, which is now known as NTPDase2, also known as CD39L1 (CD39Like1). Further human genomic analysis allowed the identification of additional members of this family and the CD39L nomenclature continued from CD39L1 to CD39L4 and at the second International Workshop of NTPDases, scientists proposed that all E-NTPDase family members can be termed as NTPDase proteins and classified in order of their discovery and characterization (Zimmermann, 2000). The four cell surface expressed NTPDases: 1, 2, 3 and 8 can be differentiated, based on their substrate specificity. They share a common feature, hydrolyzing nucleotide triphosphates, whereas they differ in nucleoside diphosphate specificity. E-NTPDase1 or CD39 hydrolyzes both nucleoside triphosphates and diphosphates. NTPDase 3 and 8 prefer ATP over ADP as substrate, whereas NTPDase2 shows a high preference for nucleoside diphosphates (Kukulski et al., 2005). NTPDase1 hydrolyzes ATP directly to AMP with minimal amounts of ADP as a transient by product, whereas during UTP hydrolysis, a significant amount of UDP is accumulated (Kukulski et al., 2005). On the other hand NTPDase2 hydrolyzes ATP to ADP, which accumulates, thus

creating agonists for P2Y1 and P2Y12 receptors (Sevigny et al., 2002). The cell surface expressing NTPDase 1, 2, 3 and 8 are anchored to the cell membrane via two transmembrane domains that are important for maintaining catalytic activity and substrate specificity, further both the N- and C-termini are intracellular. Cell surface NTPDases are considered to be most important for controlling the availability of agonists for P2 receptors. There is increasing experimental evidence that NTPDases compete with P2 receptors for the endogenously released nucleotides (Enjyoji et al., 1999; Goepfert et al., 2000), thus controlling the function

44 of P2 receptors, either by terminating or modulating their function (Alvarado-Castillo et al., 2005; He et al., 2005). Thus, NTPDases are expressed more or less in every tissue including the vasculature, immune and nervous systems having impact on several patho-physiological processes, such as cell metabolism, adhesion, migration, proliferation, differentiation, apoptosis as observed in atherosclerosis, neuro-degenerative diseases and immune rejection of transplanted organs and cells (Yegutkin, 2008).

2.4 Sympathetic Nervous System The nervous system is divided into central nervous system (CNS) and peripheral nervous system (PNS). The components of CNS are the brain and the spinal cord that are encased in bone. All the other parts of the nervous system other than the components of CNS comprise the PNS. The PNS is further divided into somatic and autonomic nervous system (Bear et al., 2007). The somatic nervous system includes the sensory and motor nerves that innervate the limbs, the joints, the muscles and the body wall. The autonomic nervous system also called the involuntary, vegetative nervous system, consists of the neurons that innervate the inner organs, blood vessels and glands, and is further divided into sympathetic and parasympathetic nervous systems (Gourine et al., 2009). The sympathetic and parasympathetic systems operate in parallel and oppose each other, and use pathways that are quite different in structure and neurotransmitters that operate (Bear et al., 2007). Both the systems have a two neuron pathway from the central nervous system to the peripheral organ. The parasympathetic nervous system is involved in various energy conserving mechanisms. The sympathetic nervous system is involved in responses that would be associated with fight or flight mechanisms, such as increasing heart rate and blood pressure as well as constricting the blood vessels in the skin and dilating them in muscles (Bear et al., 2007). The sympathetic nervous system sends its efferents to all parts of the body, which are interrupted by ganglia containing the nerve terminals of preganglionic neurons and somata of the postganglionic neurons that give rise to postganglionic axons. These postganglionic axons run in bundles into the target organs, where single axons give rise to significant branches (Boehm & Kubista, 2002). In sympathetic ganglia, preganglionic axons form synapses with the somatodendritic region of the postganglionic neurons, where one preganglionic axon innervates a number of postganglionic neurons and one postganglionic neuron receives input

45 from a number of preganglionic axons. Thus, at the sympathetic ganglia there is a neuronal convergence as well as divergence; hence it is difficult to define a specific pathway from spinal cord to the target organ (Boehm & Kubista, 2002). Postganglionic sympathetic neurons can be characterized by functional means into three different types; phasic, tonic and long-after-hyperpolarizing. Phasic neurons are characterized by the presence of M-type K+ channels, tonic by small Ca2+ activated K+ channels and long-after-hyperpolarising neurons by small and long-lasting Ca2+ activated K+ channels (Boehm & Kubista, 2002). The main transmitter released from all the preganglionic sympathetic axon terminals is acetylcholine, that depolarizes postsynaptic neurons in two phases; an early one in the millisecond range mediated by an activation of nicotinic receptors and a later one in the + range of seconds mediated by an inhibition of M-type K channels (KV7) via muscarinic receptors. ATP is co-released with acetylcholine, that might mediate excitatory synaptic transmission between sympathetic neurons (Boehm & Kubista, 2002). In most of the nerve terminals of postganglionic sympathetic axons, ATP and noradrenaline are the predominant neurotransmitters. The phenotype of a neurotransmitter is determined by the innervated target organ (Boehm, 2003b). For instance, sweat glands release a cholinergic differentiation factor that leads to the expression of choline acetyl transferase and consequently to the biosynthesis of acetylcholine in sympathetic neurons (Ernsberger & Rohrer, 1999). In addition to ATP, noradrenaline (NA) and acetylcholine (ACh), several neuropeptides are also released from postganglionic sympathetic axons and the type of peptide to be expressed in these neurons is also determined by the target organs, for example; neurons innervating sweat glands contain VIP, CGRP and/or substance P as co-transmitters to acetylcholine, whereas neurons that innervate the heart, blood vessels or the vas deferens, store and release NPY, galanin and/or somatostatin as co-transmitters to noradrenaline, that exert modulatory effects rather than being involved in ganglionic transmission (Furness et al., 1992; Elfvin et al., 1993; Benarroch, 1994; Boehm & Kubista, 2002). Thus, drugs that influence the sympathetic nervous system may exert their actions at several places within the central nervous system, sympathetic ganglia or target tissues, but the final result will always be the same: a change in the amount of sympathetic transmitters being released in effector organs (Boehm, 2003b). Thus, in vivo experiments will hardly be able to reveal whether drugs act specifically on sympathetic neurons. Hence, in vitro experiments are generally used

46 to investigate the functions of the sympathetic nervous system (Boehm, 2003b). Mechanisms of ganglionic transmission, roles of nucleotides and appropriate receptors in the signaling of sympathetic neurons, are commonly studied in primary cultures of sympathetic ganglia (Boehm, 2003b). Thus sympathetic transmitter release as the final consequence of neuronal activity within the sympathetic nervous system, one can understand that, there are two opposite effects elicited by nucleotide receptor agonists: activation of excitatory nucleotide receptors will stimulate or augment transmitter release and activation of inhibitory receptor might prevent or reduce the transmitter release. Acetylcholine and ATP are both ganglionic and sympatho-effector transmitters in the sympathetic nervous system as mentioned earlier, which exert their actions in similar ways, by activating ionotropic and metabotropic receptors. Acetylcholine and ATP cause depolarization (Connolly et al., 1993) and subsequent release of noradrenaline from cultured rat sympathetic neurons (Boehm, 1994; Boehm et al., 1995; Boehm & Kubista, 2002), by activating ionotropic nicotinic acetylcholine and P2X nucleotide receptors. Further the metabotropic actions are mediated by the muscarinic acetylcholine (mAChRs) and nucleotide P2Y receptors.

2.5 P2 receptors in sympathetic nervous system Synaptic transmission requires release of neurotransmitters from presynaptic terminals and activation of postsynaptic receptors by these neurotransmitters. Synaptic transmission can be modulated by a range a receptors and one of the most important are the P2 receptors that are known to modulate neurotransmitters release. As discussed earlier, P2Y receptors modulate different ion channels and thus affect the neurotransmitter release from the presynaptic terminal. Calcium signaling plays an important role in neuronal signaling and the P2Y

receptors such as P2Y1, 2 and 6 were shown to mediate an increase in the intracellular calcium levels in cultured mouse SCG neurons (Calvert et al., 2004). In sympathetically

innervated tissues, such as the rat vas deferens, P2Y12 and 13 receptors were shown to inhibit [3H] noradrenaline release (Queiroz et al., 2003). Likewise, in PC12 cells and rat superior

cervical ganglion (SCG) neurons, P2Y12 receptors mediate a presynaptic inhibition of noradrenaline release (Kulick & von Kugelgen, 2002; Lechner et al., 2004). Most recently,

47 P2Y12 together with P2Y1 receptors were shown to mediate autoinhibition in sympathetically innervated tissues (Quintas et al., 2009). Along the same line, evidence has been presented

that P2Y1, 12, and 13 receptors mediate inhibition of noradrenaline release in the central nervous system (Csolle et al., 2008; Heinrich et al., 2008). While most of the P2Y receptors are known to inhibit neurotransmitter release from sympathetic neurons, P2X receptors and

most probably P2X2 receptors are known to trigger noradrenaline release from cultured rat

sympathetic neurons (Boehm, 2003b). In addition P2X3 receptors were also shown to enhance noradrenaline release from rabbit ear arteries (Miyahara & Suzuki, 1987; Ishii et al., 1993; Boehm, 2003b) and saphenous arteries (Todorov et al., 1994; Boehm, 2003b), as well as in guinea pig ileum (Sperlagh et al., 2000; Boehm, 2003b). In addition to P2X receptors,

P2Y receptors most probably P2Y6 receptors were also shown to trigger noradrenaline release (Vartian et al., 2001; Boehm, 2003b).

ATP and UTP sensitive P2Y2 receptors were demonstrated to possess the capacity to produce limb muscle vasodilatation and suppress the vasoconstriction evoked by tyramine-induced release of noradrenaline at rest (Rosenmeier et al., 2008). Like in all other cells, even in sympathetic neurons P2X receptors mediate cation (Ca2+) influx which leads to depolarization and thus transmitter release (Boehm, 1999b; North, 2002; Boehm, 2003b). Several ion channels in sympathetic nervous system were shown to be

inhibited by P2Y receptors. Recently P2Y1 receptors were also shown to mediate an inhibition of Ca2+ currents in SCG neurons (Filippov et al., 2010). Almost all the known P2Y receptors heterologously expressed in rat SCG neurons were demonstrated to mediate an 2+ inhibition of N-type Ca channels: P2Y1 (Brown et al., 2000; Filippov et al., 2000), P2Y2

(Filippov et al., 1998), P2Y4 (Filippov et al., 2003), P2Y6 (Filippov et al., 1999) and P2Y12 (Simon et al., 2002) receptors. Similarly, at least two of the P2Y receptors were also suggested to mediate an inhibition of Kv7 channels in frog and rat sympathetic neurons:

P2Y4 (Meng et al., 2003) and P2Y6 (Boehm, 1998). Heterologously expressed P2Y1 receptors also mediate an inhibition of Kv7 channels in rat SCG neurons and in PC12 cells

(Brown et al., 2000; Moskvina et al., 2003). In addition, P2Y1, 2, 4 and 6 receptors expressed in rat SCG neurons were shown to mediate an inhibition of Kv7 channels (Filippov et al., 1998; 1999; Brown et al., 2000; Filippov et al., 2003). However, G protein-coupled inwardly rectifying K+ (GIRK) channels such as GIRK1 and GIRK2 (Kir3.1 and 3.2 subunits) when

48 coexpressed in rat SCG neurons with P2Y1, 4, 6 and 12 receptors were demonstrated to

activate GIRK channels. But except for P2Y12 receptors, the other P2Y receptors mentioned above showed a peculiar characteristic where, the fast activation of the channels was followed by a slower but almost a complete inactivation of the current in the continued presence of agonist (Simon et al., 2002; Filippov et al., 2004).

P2X1 receptors upon ATP stimulation mediate platelet shape change, whereas the ADP

sensitive P2Y1 and 12 receptors mediate normal platelet aggregation. P2Y1 receptors mediate

ADP-induced platelet aggregation and shape change, whereas P2Y12 receptors are

responsible for completion of the platelet aggregation response initiated by P2Y1 (Gachet, 2008). In the vasculature, different tissues/cells are known to express a variety of P2 receptors mediating vasodilatation and vasoconstriction. However on the adventitial side sympathetic nerves mediate vasoconstriction by ATP and the most important P2 receptors

here are identified to be P2X1 and P2Y receptors (Erlinge & Burnstock, 2008). P2X3 receptors of rat SCGs were suggested to be involved in the cardiac nociceptive transmission (Zhang et al., 2007). Thus, in the present study, the role of additional P2Y receptors and ion channels (M-type K+ and N-type Ca2+ channels) has been investigated for their role in noradrenaline release from the rat sympathetic neurons.

49 3 AIMS

The present study aims at investigating the roles of P2Y1 receptors and ion channels that might mediate the regulation of noradrenaline (NA) release from the rat sympathetic neurons. Precisely the following questions were addressed: • What are the additional P2Y receptors that might play a role in modulating NA release from rat SCG neurons? • What are the underlying signaling mechanisms involved in regulating the NA release from rat sympathetic neurons via P2Y receptors?

• Do P2Y1 and P2Y12 receptors interact with each other?

50 4 MATERIALS AND METHODS

4.1 Cell lines Human embryonic kidney (HEK) 293 cells, tsA 201 (a subclone of HEK 293 cells stably expressing the SV40 large T-antigen), and human astrocytoma 1321N1 cells were cultured in Dulbecco's modified Eagle's medium (PAA Laboratories, Pasching, Austria) containing 1 g l− 1 glucose and l-glutamine, supplemented with 10% fetal bovine serum (PAA) and 25,000 IU l− 1 penicillin and 25 mg l− 1 streptomycin (Sigma, Vienna, Austria). For patch- clamp experiments, cells were plated in 35 mm culture dishes coated with poly-d-lysine (PDL). For fluorescence FRET and confocal microscopy, tsA cells were plated on PDL- coated glass cover slips. PC12 cells were obtained from the European Collection of Cell Cultures (ECACC; Salisbury, UK) and kept in OptiMEM (Life Technologies, Vienna, Austria) supplemented with 0.2 mM L-glutamine (HyClone, Aalst, Belgium), 25.000 IU/l penicillin and 25 mg/l streptomycin (Sigma, Vienna, Austria), 5% fetal calf serum, and 10% horse serum (both Life Technologies, Vienna, Austria). Once per week, cell cultures were split, and the medium was exchanged twice weekly. To investigate the release of previously incorporated [3H]noradrenaline under continuous superfusion, PC12 cells were plated onto 5 mm discs, as described for the SCG neurons above. All tissue culture plastic was coated with rat tail collagen (Biomedical Technologies Inc., Stoughton, MA, USA).

PC12 cell clones stably expressing the rat P2Y1 receptor linked to the green fluorescent protein (P2Y1-GFP) were generated as described before; here, cells of clone 8 were used (Moskvina et al., 2003).

4.2 Rat primary superior cervical ganglion neurons Primary cultures of dissociated SCG neurons from neonatal rats were prepared as described before (Boehm, 1999a). Newborn Sprague-Dawley rats were sacrificed three to ten days after birth by decapitation in full accordance with all rules of the Austrian animal protection law and the Austrian animal experiment by-laws. Ganglia were removed immediately after decapitation of the animals, cut into 3 to 4 pieces, and incubated in collagenase (1.5 mg/ml; Sigma, Vienna, Austria) and dispase (3.0 mg/ml; Boehringer Mannheim, Vienna, Austria) for

51 20 min at 37 °C. Subsequently, they were further incubated in trypsin (0.25 % trypsin; Worthington, Lakewood, NJ, USA) for 15 min at 37 °C, dissociated by trituration, and resuspended in Dulbecco’s modified Eagle's Medium (InVitrogen, Lofer, Austria) containing 2.2 g/l glucose, 10 mg/l insulin, 25000 IU/l penicillin and 25 mg/l streptomycin (InVitrogen), 50 µg/l nerve growth factor (R&D Systems Inc., Minneapolis, MN, USA), and 5% fetal calf serum (InVitrogen). Finally, all cells were seeded onto 5 mm plastic discs for radiotracer release experiments and onto 35 mm culture dishes for electrophysiological experiments. All tissue culture plastic was coated with rat tail collagen (Biomedical Technologies Inc., Stoughton, MA, USA). The cultures were stored for 4 to 8 days in a humidified 5% CO2 atmosphere at 37 ºC. On days one and four after dissociation, the medium was exchanged entirely.

4.3 Transfection and generation of fluorescent tagged plasmid constructs All tsA and astrocytoma 1321N1 cells were transfected using the ExGen 500 reagent (Fermentas; St.Leon-Rot, Germany) according to the manufacturer's recommendation. 2.5 μg

DNA in total were transfected and FRET measurements were carried out 24 h later. P2X2- CFP/-YFP was a kind gift of Dr. Florentina Soto (Seattle, USA). Rat P2Y receptor fusion proteins with CFP and YFP, eNTPDase1 and eNTPDase2 with YFP were generated as described in (Schicker et al., 2009). cDNAs for rat eNTPDase1 and eNTPDase2 were kindly

provided by Herbert Zimmermann (Frankfurt, Germany), for rat P2Y1 by Georg Reiser

(Magdeburg, Germany), for rat P2Y12 by Eric Barnard (Cambridge, UK). The integrity of all generated constructs was verified by sequence analysis.

4.4 Fluorescence FRET microscopy Three filter FRET experiments (Gordon et al., 1998) were carried out in tsA 201 cells 24 h after transfection with a total of 2.5 μg DNA per cell culture with a PDL-coated cover slip, using an inverted fluorescence microscope (Zeiss), a mercury arc lamp (Zeiss HBO 100-watt intensity) and an 63× Zeiss oil immersion objective. For detection of CFP, cells were viewed with a filter set with an excitation filter of 440 nm, a dichroic beam splitter of 455 nm, and an emission filter of 480 nm. The filter set for YFP consisted of an excitation filter of 500 nm, a dichroic beam splitter of 525 nm, and an emission filter of 535 nm. The filters for FRET

52 were an excitation filter of 440 nm, a dichroic beam splitter of 455 nm, and an emission filter of 535 nm. Images were captured with a cooled CCD camera (Roper Scientific, coolsnap fx) and were analyzed with the Metamorph (Molecular Devices) and ImageJ software (http://rsbweb.nih.gov/ij/). FRET with the three-filter set system was quantified according to the method introduced by Xia and Liu (Xia & Liu, 2001) to normalize the FRET intensity to CFP and YFP concentrations in each region of interest (ROI). ROIs were selected manually and represent membranes expressing both FRET partners. For an ROI, intensity (I) from the three filter sets

was obtained after background subtraction. Then, NFRET was calculated as follows:

NFRET = (IFRET − IYFP × a − ICFP × b) / square root (IYFP × ICFP), where a = 23%, which is the percentage of CFP contribution to FRET intensity, and b = 67%, which is the percentage of YFP contribution to FRET intensity.

In each experiment, mean NFRET values of 6 to 20 ROIs were obtained, and arithmetic means of at least 5 independent experiments are shown for each data point. Analysis of statistically significant differences was performed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, and p values < 0.05 were considered indicative of statistically significant differences.

4.5 Confocal microscopy Colocalisation assays were performed on a Zeiss LSM510 confocal microscope. CFP and YFP images were captured in the multitrack mode using an argon laser (458- and 514-nm lines, respectively) and a 458/514-nm beam splitter. CFP was detected with a 475- to 525-nm band-pass filter and YFP with a long-pass 530-nm filter. The trypan blue images were captured using a helium-neon (HeNe) laser of 543 nm line. Imaging was performed with the low laser power (~ 6%) and the pinhole size of 1 - 2 µm. tsA cells grown on 14-15 mm PDL- coated cover slips were transiently transfected with plasmids encoding different CFP and YFP tagged proteins. After 24h, the cover slips were mounted on an incubation chamber, filled with 1:2 dilution of 0.05% trypan blue from a stock of 0.4% (Sigma-Aldrich) and

Krebs–Hepes buffer (10 mM Hepes, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM glucose, pH adjusted to 7.4 with NaOH). The Cells were examined with a 40X oil- immersion objective on a Zeiss LSM510 confocal microscope and the three images (CFP,

53 YFP and trypan blue) were taken simultaneously using a cooled F-View II Camera and the CellP imaging software (Olympus Soft Imaging Solutions). The images were analyzed with ImageJ software (http://rsbweb.nih.gov/ij/).

4.7 Determination of [3H]noradrenaline release [3H]noradrenaline uptake and superfusion of SCG neurons and PC12 cells was performed as described (Lechner et al., 2004). The plastic discs with dissociated neurons or PC12 cells were incubated in 0.05 μM [3H]noradrenaline ((-)-[Ring-2,5,6-3H]noradrenaline, specific activity 1.369 TBq/mmol; Perkin Elmer, Vienna, Austria) in culture medium supplemented with 1 mM ascorbic acid at 37 °C for 1 h. Thereafter, the culture discs were transferred to

small chambers and superfused with a buffer containing (mM) NaCl (120), KCl (3.0), CaCl2

(2.0), MgCl2 (2.0), glucose (20), HEPES (10), fumaric acid (0.5), Na-pyruvate (5.0), ascorbic acid (0.57), and desipramine (0.001), adjusted to pH 7.4 with NaOH. Superfusion was performed at 25 °C at a rate of about 1.0 ml/min. Collection of 4 min fractions of superfusate was started after a 60 min washout period during which excess radioactivity had been removed. Depolarization-dependent tritium overflow was triggered either by 36 monophasic rectangular electrical pulses (0.5 ms, 60 mA, 66 V/cm) delivered at 0.3 Hz or by the inclusion of 25 mM KCl (NaCl was reduced accordingly to maintain isotonicity) in the buffer for periods of 120 s. These stimulations were started after 72 (S1) and 92 minutes (S2) of superfusion. Nucleotides and nucleotide receptor agonists or antagonists were included in the buffer from minute 88 onward (see figure 5). Tetrodotoxin (TTX), whenever appropriate, was included in the buffer after 50 min of superfusion (i.e. 10 min prior to the start of sample collection). The radioactivity remaining in the cells after the completion of experiments was extracted by immersion of the discs in 2 % (v/v) perchloric acid followed by sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting (Packard Tri-Carb 2800 TR) with a counting efficiency of 63%. Radioactivity released in response to electrical field stimulation from rat sympathetic neurons after labelling with tritiated noradrenaline under conditions similar to those of the present study had previously been shown to consist predominantly of the authentic transmitter and to contain only small amounts (<15%) of metabolites (Schwartz & Malik, 1993). Hence, the

54 outflow of tritium measured in this study was assumed to reflect the release of noradrenaline and not that of metabolites. The spontaneous (unstimulated) rate of [3H] efflux was obtained by expressing the radioactivity retrieved during a collection period as percentage of the total radioactivity in the cultures at the beginning of this period. Stimulation-evoked tritium overflow was calculated as the difference between the total tritium outflow during and after stimulation and the estimated basal outflow which was assumed to follow a linear time course throughout experiments. Therefore, basal outflow during periods of stimulation was assumed to equate to the arithmetic mean of the samples preceding and those following stimulation, respectively. Differences between total and estimated basal outflow during periods of stimulation were expressed as percentages of total radioactivity in the cultures at the onset of stimulation (% of total radioactivity; S%). The amount of radioactivity in the cultures at the beginning of each collection period is calculated by summing up the radioactivity remaining in the cells at the end of experiments and that retrieved during the respective and all subsequent collection periods. As the amount of depolarization- or drug-induced tritium overflow may vary considerably between different cultures (Scholze et al., 2002), the effects of nucleotides on depolarization- dependent release were evaluated by determining changes in the ratio of tritium overflow evoked during the two periods of electrical or K+ stimulation (S2/S1). When cultures had been subjected to a certain treatment (e.g. cholera toxin or U73122), control experiments were also performed in sister cultures that had not been exposed to that treatment (i.e. remained ‘untreated’). In order to directly compare the effects of ADP observed in the absence with those observed in the presence of antagonists, ADP was applied either alone or in combination with the appropriate antagonist. As controls, either the antagonist alone or solvent were applied. Thereafter, the S2/S1 ratio obtained with ADP was expressed as percentage of the corresponding S2/S1 value obtained in its absence (S2/S1, % of control).

4.8 Electrophysiology

Currents through KV7 channels in SCG neurons and in tSA cells expressing KV7 channel

subunits, so called M currents (IM), were determined using the perforated patch clamp technique as described (Lechner et al., 2003). Currents were recorded at room temperature

55 (20-24°C) from single SCG neurons in vitro and from single tsA cells using an Axopatch 200B amplifier and the pCLAMP 8.0 hard- and software (Molecular Devices, Sunnyvale, CA, USA). Signals were low-pass filtered at 5 kHz, digitized at 10 to 50 kHz, and stored on an IBM compatible computer. Traces were analyzed off-line by the Clampfit 8.1 program (Molecular Devices). Patch electrodes were pulled (Flaming-Brown puller, Sutter Instruments, Novato, CA, USA) from borosilicate glass capillaries (Science Products,

Frankfurt/Main, Germany), front-filled with a solution consisting of (mM) K2SO4 (75), KCl

(55), MgCl2 (8), and HEPES (10), adjusted to pH 7.3 with KOH. Electrodes were then backfilled with the same solution containing 200 μg/ml amphotericin B (in 0.8 % DMSO) which yielded tip resistances of 2 to 3 MΩ. The bath solution contained (mM) NaCl (140),

KCl (3.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20), HEPES (10), adjusted to pH 7.4 with NaOH. Tetrodotoxin (TTX; 0.5 μM) was included to suppress voltage-activated Na+ currents. ADP and all other drugs were applied via a DAD-12 drug application device (Adams & List, Westbury, NY, USA) which permits a complete exchange of solutions surrounding the cells under investigation within less than 100 ms (Boehm, 1999b). To

investigate IM, cells were held at a potential of -30 mV, and thrice per minute 1 s

hyperpolarisations to -55 mV were applied to deactivate the KV7 channels; the difference between current amplitudes 20 ms after the onset of hyperpolarisations and 20 ms prior to re-

depolarisation was taken as a measure for IM. Amplitudes obtained during the application of test drugs (b) were compared with those measured before (a) and after (c) application of these drugs by calculating 200b / (a+c) = % of control or 100 - (200b/[a+c]) = % inhibition (Boehm, 1998).

56 5 RESULTS 5.1 Enhancement of stimulation-evoked noradrenaline release from SCG neurons treated with pertussis toxin by ADP ADP, at a concentration of 100 μM, has been found to reduce noradrenaline release from rat SCG neurons triggered by 30 mM K+; however, in neurons treated with pertussis toxin to prevent the signalling via inhibitory G proteins, the nucleotide rather tended to enhance stimulation-evoked release (Lechner et al., 2004). Therefore, the effect of ADP was investigated in SCG cultures treated with pertussis toxin (100 ng/ml) for 24 hours and labeled with [3H]noradrenaline. In these experiments, 100 μM ADP clearly enhanced tritium overflow triggered by electrical field stimulation, but left spontaneous [3H] outflow unaltered (figure 6A and B). The lack of change in spontaneous outflow suggests that ADP did not trigger action potential-dependent exocytosis, as does the activation of other Gq coupled receptors, such as B2 bradykinin (Scholze et al., 2002) or M1 muscarinic acetylcholine receptors (Lechner et al., 2003). To confirm that the effect of ADP was not due to enhanced action potential firing, experiments were repeated in the presence of the Na+ channel blocker TTX (0.1 μM). As TTX prevents electrically evoked noradrenaline release from SCG neurons (Boehm, 1999b), tritium overflow was stimulated by exposing the cultures to 25 mM KCl for 2 minutes. Under these conditions, ADP also raised stimulation-evoked tritium overflow (figure 6 C and D) and left the spontaneous outflow unaltered. Thus, the facilitation by ADP does not require action potential propagation.

57

Figure 6 Enhancement of [3H]noradrenaline release from rat SCG neurons treated with pertussis toxin by ADP. SCG cell cultures were treated with PTX (100 ng/mL for 24 h), were labelled with [3H]noradrenaline and superfused. When appropriate (C and D), 0.1 µM TTX were present from min 50 of superfusion onward. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected. Tritium overflow was stimulated twice (S1 after 72 min and S2 after 92 min of superfusion) by 2 min exposures to either electrical pulses (A and B) or 25 mM KCl (C and D). Either 100 µM of ADP or the appropriate solvent were present from minute 88 onward, as indicated by the arrows in A and C. A and C show exemplary time courses of fractional [3H] outflow as a percentage of the total radioactivity in the cells (n = 3). B and D display the S2/S1 ratios of tritium overflow evoked by electrical stimulation (B) or 25 mM K+ (D) in the presence of either solvent or 100 µM ADP (n = 11- 12); the p values for the statistical significance of differences (unpaired Students t-test) are indicated above the bars.

58 5.2 The enhancement of stimulation-evoked noradrenaline release from pertussis toxin-

treated SCG neurons is mediated by P2Y1 receptors

Amongst the P2Y receptors, P2Y1, P2Y12, and P2Y13 are the primary binding sites for ADP (von Kugelgen, 2006). To differentiate between these three, ADP, ATP , and 2-MeSATP

were chosen as agonists, the latter being a preferred agonist of P2Y1, but not of P2Y12 and

P2Y13, receptors of the rat (von Kugelgen, 2006). All these nucleotides proved to enhance electrically evoked [3H] overflow from cultures treated with pertussis toxin; in the resulting concentration-response curves, 2-MeSATP was more potent than ADP and ATP, which were about equipotent (figure 7A). When considering the effects of ATP at concentrations higher than 3 µM, one must not forget the concomitant activation of P2X receptors which also leads to noradrenaline release from SCG neurons (Boehm, 1999b). Nevertheless, the rank order of

agonist potency 2-MeSATP > ATP = ADP argues for a role of P2Y1 receptors, even though rather high concentrations of these nucleotides were required. To corroborate the results obtained with agonistic nucleotides, suramin, reactive blue 2 and MRS 2179 were employed as antagonists and applied together with ADP. While the former two block all three ADP-sensitive P2Y receptors (von Kugelgen, 2006), MRS 2179 is

selective for P2Y1 (Boyer et al., 1998). All three antagonists abolished the facilitation of tritium overflow by ADP (Figure 7B), thereby confirming that this effect was mediated by

P2Y1 receptors.

59 Figure 7 Pharmacological characterization of the receptor mediating the enhancement of [3H]noradrenaline release. SCG cell cultures were treated with PTX (100 ng/mL for 24 h), were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected, and tritium overflow was evoked by electrical field stimulation as shown in figure 5A. A shows the concentration-dependent increase in the S2/S1 ratio of tritium overflow caused by ADP, ATP or 2-MesATP (n = 6 to 13). Nucleotides used at the µM concentrations indicated or the appropriate solvents were present from minute 88 onwards. B shows the increase in the S2/S1 ratio caused by the indicated concentrations of ADP as percentage of control in the absence or presence of the indicated concentrations (in µM) of suramin (n = 8-9), reactive blue 2 (RB2; n = 10-12), or MRS 2179 (n = 8-9). P values for the significance of differences between the results obtained in the absence and presence of antagonists are indicated above the bars.

5.3 Enhancement of stimulation-evoked noradrenaline release from SCG neurons not treated with pertussis toxin The above data indicate that ADP has the ability of enhancing stimulation-evoked noradrenaline release when the signalling via inhibitory G proteins is blocked by pertussis toxin. To reveal whether the facilitation by ADP may also occur in neurons with functional Gi/o proteins, experiments were repeated in cultures not treated with PTX; there, 100 μM ADP caused a significant reduction of electrically evoked tritium overflow (figure 8A) as

described before; this inhibition of noradrenaline release was suggested to involve P2Y12 receptors (Lechner et al., 2004). Therefore, experiments were performed in the presence of

the P2Y12 antagonist cangrelor (Jacobson & Boeynaems, 2010). Cangrelor (10 μM), when applied alone, did not cause obvious changes in tritium outflow (figure 8C). However, when ADP was used together with cangrelor, the nucleotide caused a significant increase in tritium overflow (figure 8 C and D). Hence, the facilitation by ADP can be observed as soon as

P2Y12 receptors are blocked.

To reveal whether this facilitatory effect of ADP is mediated by P2Y1 receptors, the selective

and potent P2Y1 receptor agonist MRS2365 (Chhatriwala et al., 2004) was employed instead of ADP. At a concentration of 0.1 μM, MRS2365 significantly enhanced electrically evoked tritium overflow (figure 8 E and F). Thus, the facilitation of tritium overflow can also be seen

60 in cultures not treated with pertussis toxin if either the P2Y12 antagonist cangrelor or the

selective P2Y1 agonist MRS2365 is used. Nevertheless, all future experiments were performed in cultures treated with pertussis toxin to avoid excessive use of these specific P2Y receptor ligands.

Figure 8 Modulation of [3H]noradrenaline release from SCG neurons by ADP, MRS2365 and cangrelor. SCG cell cultures were labelled with [3H]noradrenaline and

61 superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected, and tritium overflow was evoked by electrical field stimulation as shown in figure 1A. A, C, and E show exemplary time courses of fractional [3H] outflow as a percentage of the total radioactivity in the cells (n = 3); 100 μM ADP, 10 μM cangrelor, 0.1 μM MRS 2365, or the appropriate solvent were present from minute 88 onward as indicated by the arrows. B shows S2/S1 ratios obtained in the presence of either solvent or 100 μM ADP (n = 11). D shows S2/S1 ratios obtained in the presence of either 10 μM cangrelor or 10 μM cangrelor plus 100 μM ADP (n = 6). F shows S2/S1 ratios obtained in the presence of either solvent or 0.1 μM MRS 2365 (n = 12); the p values for the statistical significances of differences (unpaired Students t-test) are indicated above the bars.

5.4 The enhancement of stimulation-evoked noradrenaline release from SCG neurons by ADP involves phospholipase C As the facilitation by ADP was observed in cultures treated with PTX, this effect cannot be mediated by inhibitory G proteins. However, the facilitation of noradrenaline release via presynaptic GPCRs may involve stimulatory Gs proteins (Kubista & Boehm, 2006). To test for this alternative, cultures were treated not only with PTX, but also with cholera toxin (100

ng/ml), for 24 hours; this manipulation eliminates αs G protein subunits from primary cultures of sympathetic neurons (Boehm et al., 1996). However, the facilitation of electrically evoked [3H] overflow was the same whether cultures had been treated with PTX only, or with PTX plus cholera toxin (Figure 9A). Thus, the effects of ADP do not involve Gs proteins.

P2Y1 receptors are most commonly linked to proteins of the Gq family and thereby to phospholipase C (Abbracchio et al., 2006). To test for a role of these latter enzymes, cultures were treated with 3 µM U73122 which irreversibly blocks signalling via phospholipase C in SCG neurons (Bofill-Cardona et al., 2000). In neurons treated with U73122 and PTX, ADP failed to significantly enhance electrically evoked tritium overflow. However, in sister cultures treated with PTX only, ADP clearly caused facilitation (Figure 9B). Thus, the facilitatory effects of the nucleotide involve an activation of phospholipase C.

62

Figure 9 The enhancement of [3H]noradrenaline release from rat SCG neurons by ADP involves phospholipase C, but not Gs proteins. All SCG cell cultures were treated with PTX (100 ng/mL for 24 h). In addition, some cultures were treated with cholera toxin or remained otherwise untreated. Thereafter, the cultures were labelled with [3H]noradrenaline in the absence (untreated) or presence of 3 µM U73122 and were then superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected, tritium overflow was evoked by electrical field stimulation, and ADP was applied as shown in figure 5A. A shows the S2/S1 ratios of tritium overflow in the absence (control) or presence of 100 µM ADP in either untreated or cholera toxin-treated neurons (n = 10-12). B shows the S2/S1 ratios of tritium overflow in the absence or presence of 100 µM ADP in either untreated or U73122-treated neurons (n = 9). P values for the significance of differences between the results obtained in the absence and presence of ADP are indicated above the bars; * indicates a significant difference versus the corresponding result obtained in untreated cultures at p < 0.05; ns indicates no such significance.

5.5 The enhancement of stimulation-evoked noradrenaline release from SCG neurons does not involve protein kinase C or increases in intracellular Ca2+ Activation of presynaptic receptors linked to phospholipase C can lead to facilitation of noradrenaline release through increases in intracellular Ca2+ and subsequent activation of protein kinase C (Kubista & Boehm, 2006). To test for a role of the latter mechanism, experiments were performed in the presence and absence of 10 µM H-7, an inhibitor of protein kinases A, C, and G, with affinities for these enzymes in the low micromolar range

63 (Hidaka et al., 1984). However, the facilitation of electrically evoked release was the same in the absence and presence of this broad spectrum kinase inhibitor (Figure 10A). Increases in intracellular Ca2+ may facilitate transmitter release independently of protein kinases (Kubista & Boehm, 2006). Therefore, the intracellular Ca2+ stores of the SCG neurons were depleted by the Ca2+-ATPase inhibitor thapsigargin (Bofill-Cardona et al., 2000), and the facilitatory effect of ADP was assessed again. However, the facilitation of electrically evoked [3H] overflow by ADP was not different whether 0.3 µM thapsigargin was present or not (Figure 10B). Hence, the effect of ADP is independent of increases in intracellular Ca2+ and activation of protein kinases A, C, and G.

Figure 10 The enhancement of [3H]noradrenaline release from rat SCG neurons by ADP is not altered by inhibitors of protein kinase C or Ca2+-ATPase. SCG cell cultures were treated with PTX (100 ng/mL for 24 h), were labelled with [3H]noradrenaline and superfused. When appropriate, 10 µM H-7, 0.3 µm thapsigargin, or 0.1 % DMSO was present from min 50 of superfusion onward. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected, tritium overflow was evoked by electrical field stimulation, and ADP was applied as shown in figure 5A. A shows the S2/S1 ratios of tritium overflow in the absence (control) or presence of 100 µM ADP applied either in a solution containing DMSO, or in a solution containing H-7 (n = 9). B shows the S2/S1 ratios of tritium overflow in the absence (control) or presence of 100 µM ADP applied either in a solution containing DMSO or in a solution containing thapsigargin (n = 9). P values for the significance of differences between the results obtained in the absence and presence of ADP

64 are indicated above the bars; ns indicates no significant difference versus the corresponding result obtained in the presence of DMSO.

5.6 Recombinant P2Y1 receptors mediate enhancement of stimulation-evoked noradrenaline release from PC12 cells treated with pertussis toxin.

PC12 cells, in contrast to SCG neurons, do not express endogenous P2Y1 receptors

(Moskvina et al., 2003), but both types of cells do express P2Y12 and P2Y13 receptors

(Lechner et al., 2004). To investigate whether P2Y1 receptors might mediate an enhancement of transmitter release in another neuronal background than SCG neurons, either

nontransfected PC12 cells or a PC12 cell clone stably expressing rat P2Y1-GFP (Moskvina et al., 2003) were compared with respect to the modulation of stimulation-evoked noradrenaline release by ADP. In agreement with previous results (Lechner et al., 2004), ADP (10 μM) reduced K+-evoked tritium overflow from non-transfected PC12 cells by about 50 %; however, when these cells had been treated with pertussis toxin (100 ng/ml for 24 h), ADP

failed to cause any significant change (figure 11A). In PC12 cells expressing P2Y1 receptors, for comparison, 10 μM ADP reduced [3H] overflow by only 15% and when the cells had been exposed to pertussis toxin ADP caused a significant enhancement of K+-evoked

overflow (figure 11B). Thus, the expression of P2Y1 receptors in PC12 cells is sufficient to counteract the inhibition of transmitter release by ADP and to mediate an ADP-dependent enhancement of release when cells had been treated with pertussis toxin.

65 3 Figure 11 Enhancement of [ H]noradrenaline release from PC12 cell expressing P2Y1 receptors by ADP. PC12 cell cultures were treated with PTX (100 ng/ml for 24 h) or remained untreated, were labelled with [3H]noradrenaline and superfused. Subsequent to a 60 min washout period, 4 min fractions of superfusate were collected. Tritium overflow was stimulated twice (S1 after 72 min and S2 after 92 min of superfusion) by 2 min exposures 25 mM KCl. ADP (10 μM) was applied as shown in figure 5B. A shows the S2/S1 ratios of tritium overflow from non transfected (wild type; wt) PC12 cells in the absence (control) or presence of 10 μM ADP (n = 6). B shows the S2/S1 ratios of tritium overflow from PC12

cells expressing rat P2Y1-GFP in the absence (control) or presence of 10 μM ADP (n = 6). P values for the significance of differences between the results obtained in the absence and presence of ADP are indicated above the bars; ns indicates a lack of significant difference.

5.7 P2Y1 receptors mediate inhibition of KV7 channels by ADP

Recombinant P2Y1 receptors mediate an inhibition of currents through KV7 channels (IM) in

PC12 cells (Moskvina et al., 2003). Moreover, ADP and UDP had been found to inhibit IM in

rat SCG neurons, but this effect was suggested to involve P2Y6 receptors (Boehm, 1998).

Most recently, evidence has been presented for an inhibition of KV7 channels of SCG

neurons via P2Y1 receptors (Filippov et al., 2010). Here, we re-evaluated the receptors

mediating the inhibition of KV7 channels by ADP in SCG neurons. In line with our previous

results, 10 µM ADP reduced IM relaxation amplitudes, and this effect was entirely reversible

(Figure 12A). To reveal whether P2Y1 receptors might mediate this effect, ADP was also applied in the presence of MRS 2179 which abolished the inhibition by ADP (Figure 12 A and C). To verify, that the antagonism by MRS 2179 (30 µM) was specific for the action of

ADP, 10 µM UDP was also used to inhibit IM; this latter effect, however, remained unaltered in the presence of MRS 2179 (Figure 12D).

To confirm the results obtained with the P2Y1 antagonist, the specific P2Y1 agonist MRS 2365 (Chhatriwala et al., 2004) was employed again. As expected, 0.1 µM MRS 2365 also

reduced IM in an entirely reversible manner (Figure 12B), and the inhibition was the same as

that by 10 µM ADP (Figure 12E). Thus, in rat SCG neurons P2Y1 receptors, in addition to

P2Y6, mediate an inhibition of KV7 channels.

66

Figure 12 P2Y1 and P2Y6 receptors mediate an inhibition of currents through KV7

channels (IM) in SCG neurons. Currents were activated at -30 mV and quantified by determining the amplitudes of the slow current deactivation relaxations during 1s

hyperpolarizing voltage steps to -55 mV. These IM amplitudes were determined in the presence of ADP (10 µM), UDP (10 µM), or MRS2365 (0.1 µM) applied either alone or together with MRS2179 (30 µM). A shows original current traces recorded from one neuron before, during and after the exposure to ADP or ADP plus MRS2179. B shows original current traces recorded from another neuron before, during and after the exposure to

MRS2365. C summarizes the inhibition of IM by ADP in the absence or presence of

MRS2179 (n = 9). D summarizes the inhibition of IM by UDP in the absence or presence of

MRS2179 (n = 10). E summarizes the inhibition of IM by ADP, UDP, or MRS2365 (n = 6

67 to12). P values for the significance of differences between the results obtained in the absence and presence of MRS2179 are indicated above the bars; **, *** indicate significant differences versus the inhibition by UDP at p < 0.01 and p < 0.001, respectively.

5.8 Validation of FRET measurements by well documented interactions It is already known that different P2Y receptors, ectoenzymes and ion channels are expressed on presynaptic nerve terminals of rat SCG neurons (Moskvina et al., 2003; Lechner et al., 2004), thus it would be of great interest to study their interactions. Thus the most popular method to study membrane protein interactions is FRET and to learn whether fusion of fluorescent proteins to the N-terminus of NTPDases and C-terminus of P2Y receptors might influence membrane targeting or functions of the respective enzymes and receptors. They were investigated either with the malachite green phosphatase assay or by electrophysiology and the results indicated that, the fluorescent ectonucleotidases and the P2 receptors studied were targeted to the membrane as their unmodified counterparts and operated in a comparable manner (Schicker et al., 2009). Although FRET measurements are very specific in terms of determining distances between adjacent fluorescent proteins, the differences in arbitrary fluorescence values are small and may be affected by systematic errors (Pfleger & Eidne, 2005). Therefore, the system has been gauged by comparison of the present results with previously published positive and negative controls. As first example, the human serotonin transporter (SERT) has been used, which homooligomerizes in plasma membranes; the specificity of this interaction was

documented by showing that the human SERT did not oligomerize with human D2 dopamine

receptors (Schmid et al., 2001). Accordingly, high NFRET values were obtained when CFP- and YFP-tagged SERTs were co-expressed in HEK 293 cells (0.176 ± 0.017; n = 3). In

contrast, the NFRET values obtained after co-expression of YFP-tagged D2 receptors and CFP- tagged SERTs (0.077 ± 0.014; n = 3) were indistinguishable from the values obtained after co-expression of the two fluorescent moieties only (0.063 ± 0.013; n = 7) (Figure 13D). Nevertheless, in overlays of the fluorescent micrographs obtained with wavelengths exciting either CFP or YFP, an unequivocal co-localization of either the two differently tagged

SERTs or CFP-SERT and YFP-D2 within the plasma membrane was observed (Figure 13A) (Schicker et al., 2009). Hence, all the fluorescent proteins were present in the membrane, but

68 only the interacting ones gave high NFRET values. To further validate the NFRET value

determinations, YFP-D2 was expressed together with a CFP-tagged human metabotropic

glutamate receptor 5 (mGluR5). Again, the NFRET values (0.081 ± 0.009; n = 3) were not different from those obtained with the two fluorescent moieties only. In contrast, co-

expression of CFP- and YFP-tagged versions of mGluR5 resulted in NFRET values (0.175 ± 0.031; n = 3) as high as those obtained with the two differently tagged SERTs (Figure 13D). These results are in line with previous FRET experiments and confirm that mGluR5 is able to homo-oligomerize in the plasma membrane, but unable to closely

associate with D2 receptors (Calo et al., 2005). Amongst the purinergic receptors, P2X receptor subunits are known to form homomeric or

heteromeric trimers (Khakh & North, 2006), and direct contact between P2X2 subunits in the plasma membrane has been documented via FRET microscopy (Fisher et al., 2004).

Although the functional experiments clearly indicated that P2X2-YFP is targeted to the

membrane, first the co-localization of the two differently labeled P2X2 subunits in the plasma membrane by confocal laser microscopy has been established (Figure 13B). Then, fluorescence micrographs were taken with filter combinations to selectively visualize the localization of CFP, YFP, or the potentially occurring FRET interaction (Figure 13C). Finally, using the fluorescent CFP- and YFP-micrographs corrected for the bleed-through

errors, NFRET values were calculated for co-expressed CFP- and YFP-tagged P2X2 subunits as determined in membrane regions (0.183 ± 0.008; n = 7) were in the same range as those for the differently tagged SERTs and mGluRs (Figure 13D).

P2X2 subunit containing receptors are mainly expressed in the nervous system (Khakh & North, 2006), and their proper function relies on the presence of NTPDase1 (Schaefer et al.,

2007). Therefore, YFP-tagged NTPDase1 with CFP-tagged P2X2 subunits were co-expressed

in tsA cells and studied, which might result in NFRET values indicative of a close association of these two proteins in the plasma membrane. However, the values were not different from those obtained with the two fluorescent moieties only and were in the same range as those for

YFP-D2 plus CFP-SERT and YFP-D2 plus CFP-mGluR5, respectively (Figure 13D)

(Schicker et al., 2009). Nevertheless, YFP-tagged NTPDase1 and CFP-tagged P2X2 subunits

69 were co-localized in the plasma membrane as evidenced by confocal microscopy (Figure

13B), but gave no positive FRET signals in the calculated NFRET values (Figure 13D).

A B

C

P2X2-C P2X2-Y Overlay

P2X2-C NTPD1-Y Overlay

D

Figure 13 Validation of FRET measurements by well documented interactions. A shows pseudocolour representations of fluorescent micrographs of the human serotonin transporter

70 fused to CFP (SERT-C) or YFP (SERT-Y) and the human D2 dopamine receptor fused to YFP (D2-Y). The right hand micrographs show overlays of the two pictures to the left. B shows pseudocolour representations of fluorescent confocal laser scanning micrographs of the P2X2 subunit fused to CFP (P2X2-C) or YFP (P2X2-Y) and NTPDase1 fused to YFP (NTPD1-Y). In addition, a membrane staining by trypan blue is depicted. The right hand micrographs show overlays of the three pictures to the left. Note the white lining of the membrane region where the subsequent FRET measurements were performed. C shows pseudocolour representations of fluorescent micrographs of the same fusion proteins as in B.

Figure D taken from Schicker et al 2009, depicts NFRET values obtained with the fusion proteins mentioned above and with mGluR5 fused to CFP (mGluR5-C) or YFP (mGluR5-Y), YFP-tagged NTPDase2 (NTPD2-Y), and CFP or YFP. and indicate significant differences versus the values obtained with CFP and YFP at p < 0.01 and p < 0.001, respectively (n = 3 to 8).

For comparison, analogous experiments were carried out with YFP-tagged NTPDase2, and

the results were comparable with those for NTPDase1 plus P2X2 (Figure 13D) (Schicker et

al., 2009). In all future FRET experiments, the combinations P2X2-C plus P2X2-Y and P2X2- C plus NTPD1-Y were used for statistical comparisons as prototypic examples of membrane

protein pairs that give high and low NFRET values, respectively.

5.10 Specific interactions between P2Y1 and P2Y12 receptors Earlier results confirmed that two different P2Y receptors modulated noradrenaline release

from rat sympathetic neurons: P2Y12 receptors inhibited noradrenaline release (Lechner et al.,

2004) and P2Y1 receptors facilitated noradrenaline release (results shown above). As the two different P2Y receptors are expressed on the same nerve terminal modulating noradrenaline release, thus next step would be to evaluate whether these two P2Y receptors are able to closely associate with each other in cell membranes in a homo- or heteromeric manner. Thus, fluorescently tagged versions of these two P2Y receptors were expressed in a pair wise manner. For both these proteins the proper insertion in the membrane has been evidenced by functional experiments in previous experiments (Schicker et al., 2009).

71 The NFRET values obtained for the pairs of fluorescently labeled P2Y1 and P2Y12 receptors

were significantly different from the results obtained with P2X2-C plus NTPD1-Y in parallel

experiments, but not from those obtained with P2X2-C plus P2X2-Y (Figure 14A). Moreover,

co expression of fusion proteins of P2Y1 receptor with NTPDase2 resulted in NFRET values

that were not significantly different from negative control (P2X2-C plus NTPD1-Y). Further,

the NFRET values of P2Y12-C expressed together with P2Y12-Y and P2Y1-Y and P2Y1-C

expressed together with P2Y1-Y were significantly different from the negative (P2X2-C plus

NTPD1-Y), but not from the positive (P2X2-C plus P2X2-Y) controls. Thus, the GPCRs for adenine nucleotides displayed propensities to homo and heterooligomerize in the membranes.

A

B

NFRET

72 Figure: 14 Interactions between P2Y1 and P2Y12 receptors revealed by FRET microscopy.

A depicts NFRET values obtained with either CFP or YFP tagged P2Y1 (P2Y1-C and P2Y1-

Y), P2Y12 (P2Y12-C and P2Y12-Y), NTPDase1 (NTPD1-Y) and NTPDase2 (NTPD2-Y) as shown in the figure. indicates significant differences versus the values obtained with P2X2-C plus NTPD1-Y at p < 0.001 (n = 10 to 15), respectively; ns indicates the lack of

such a significance. B depicts NFRET values obtained with P2Y1 receptors fused to CFP (P2Y1-C) and YFP (P2Y1-Y) and also with NTPDase2 fused to YFP (NTPD2-Y). In

addition, unlabelled P2Y1 or NTPDase2 were expressed together with these fusion proteins where indicated. indicates significant differences versus the values obtained with P2Y1-

C plus P2Y1-Y at p < 0.001 (n = 10 to 15); ns indicates the lack of such a significance.

To confirm that interactions observed by FRET microscopy as described above are specific,

competition experiments were performed challenging the homomeric interaction of P2Y1

receptors. When P2Y1-C and P2Y1-Y were coexpressed together with non-tagged P2Y1, the

NFRET values were significantly reduced. When these experiments were repeated with non

fluorescent NTPDase2 instead of P2Y1, however, there was no significant change in the

NFRET values for coexpressed P2Y1-C and P2Y1-Y (Figure 14B). Hence, interactions revealed by FRET microscopy as above, can only be disrupted by appropriate interaction partners, but

not by membrane proteins that were not interacting according to the NFRET values obtained.

73 6 DISCUSSION

Presynaptic facilitation by P2Y1 receptors

P2Y1 receptors are widely distributed in the central and peripheral nervous system and mediate a plethora of effects including the modulation of voltage- and transmitter-gated ion

channels (Hussl & Boehm, 2006). However, evidence for presynaptic P2Y1 receptors is

scarce and, if available, only in favour of inhibitory presynaptic P2Y1 receptors, as suggested for peripheral sensory neurons (Gerevich et al., 2004), for hippocampal neurons (Rodrigues et al., 2005; Csolle et al., 2008), for spinal cord neurons (Heinrich et al., 2008), and also for sympathetic neurons (Quintas et al., 2009). In general, presynaptic P2Y receptors have been known to mediate inhibition, but not facilitation, of transmitter release (Goncalves & Queiroz, 2008). Hence, the results of this study provide the first evidence for an enhancement

of transmitter release via presynaptic P2Y1 receptors, thereby delivering a showcase of facilitatory presynaptic P2Y receptors. In postganglionic sympathetic neurons, multifarious evidence for inhibitory and facilitatory presynaptic P2 receptors has been presented. The pharmacological data support the idea that the nucleotide-dependent presynaptic facilitation involves P2X receptors, whereas the inhibition involves P2Y receptors (Boehm & Kubista, 2002; Sperlagh et al., 2007), most

likely P2Y12 (Queiroz et al., 2003; Lechner et al., 2004). This was corroborated in the present study, as ADP caused an inhibition of electrically evoked noradrenaline release from SCG

neurons which was reverted into facilitation by the P2Y12 antagonist cangrelor (Jacobson & Boeynaems, 2010). Likewise, in SCG neurons treated with pertussis toxin to inactivate the

signalling cascades of inhibitory P2Y12 and P2Y13 receptors, ADP also enhanced electrically evoked noradrenaline release. Moreover, this effect was observed when release was triggered by depolarizing K+ concentrations in the presence of TTX in order to block action potential propagation. Thus, the site of action for the facilitation of transmitter release by ADP must be in close proximity to the sites of vesicle exocytosis, i.e. must be a bona fide presynaptic receptor. The presynaptic receptor mediating the facilitation of noradrenaline release was a P2Y

receptor, more precisely a P2Y1 receptor, as indicated by the following results: (i) 2-

MesATP, an agonist at rat P2Y1 receptors (Dixon, 2000) enhanced stimulation-evoked

74 noradrenaline release from SCG neurons treated with pertussis toxin; (ii) 2-MesATP was

more potent than ADP as previously demonstrated for rat P2Y1 receptors (Vohringer et al., 2000); (iii) the facilitation by ADP was abolished by suramin and reactive blue 2, which are

both known as P2Y1 antagonists (von Kugelgen, 2006); (iv) the selective P2Y1 antagonist MRS 2179 (Boyer et al., 1998) also abolished the facilitatory affect of ADP; (v) finally, the

specific P2Y1 agonist MRS 2365 (Chhatriwala et al., 2004) enhanced stimulation-evoked noradrenaline release even when the signalling cascades of inhibitory P2Y receptors had not been impaired by pertussis toxin.

The capability of P2Y1 receptors to mediate facilitation of transmitter release was also confirmed using recombinant receptors. In PC12 cells, the activation of heterologously + expressed rat P2Y1 receptors led to an increase in K -evoked noradrenaline release when signalling via inhibitory GPCRs was prevented by pertussis toxin; this effect was not

observed in the absence of P2Y1 receptors. In PC12 cells expressing P2Y1 receptors but not treated with the bacterial toxin, ADP caused a reduction of depolarization-evoked noradrenaline release as it did in non transfected PC12 cells. However, this inhibitory effect of ADP was much more pronounced in non transfected PC12 cells than in cells expressing

P2Y1 receptors. This indicates that ADP simultaneously activates the facilitatory P2Y1

receptors and inhibitory P2Y12 or possibly P2Y13 receptors.

P2Y1 receptors are most commonly linked to phospholipase C via proteins of the Gq family (Abbracchio et al., 2006). In accordance with this notion, the PLC inhibitor U73122 abolished the facilitation by ADP, but removal of Gs proteins by a cholera toxin treatment had no such effect. Activated PLC employs membrane phosphatidylinositol 4,5- bisphosphates to generate inositol trisphosphate and diacylglycerol which then mediate increases in intracellular Ca2+ and activation of protein kinase C, respectively (Suh & Hille, 2007). However, none of these effects was involved in the facilitation of noradrenaline 2+ release via presynaptic P2Y1 receptors, as neither the depletion of intracellular Ca stores, nor the inhibition of a set of protein kinases including protein kinase C were sufficient to prevent this facilitation.

Heterologously expressed P2Y1 receptors mediate an inhibition of Kv7 channels in SCG neurons (Brown et al., 2000) as well as in PC12 cells (Moskvina et al., 2003). Moreover,

75 ADP has been found to inhibit Kv7 channels in SCG neurons, but it has been concluded that

this effect was mediated by endogenously expressed P2Y6 receptors (Boehm, 1998). Rat

SCG neurons are known to express endogenous P2Y1, 2, 4, 12, and 13 in addition to P2Y6 receptors (Moskvina et al., 2003; Lechner & Boehm, 2004). The present results clearly show

that endogenous P2Y1 receptors contribute to the regulation of Kv7 channels in SCG neurons

by nucleotides, as the inhibition by ADP was abolished by the selective P2Y1 antagonist

MRS 2179 (Boyer et al., 1998) and mimicked by the specific P2Y1 agonist MRS 2365

(Chhatriwala et al., 2004). The inhibition of IM by UDP, in contrast, was not altered by MRS

2179, thus indicating that P2Y1 and P2Y6 receptors can control Kv7 channels independently of each other. While this work was in progress, the inhibition of Kv7 channels in rat SCG

neurons by MRS 2365 via endogenous P2Y1 receptors has been reported by others (Filippov et al., 2010). Activation of presynaptic Kv7 channels leads to a decrease in transmitter release from cerebrocortical nerve terminals, while inhibition causes the opposite effect (Luisi et al., 2009), and the same holds true for SCG neurons (Hernandez et al., 2008). Moreover, presynaptic muscarinic receptors have been shown to facilitate transmitter release through an inhibition of Kv7 channels (Martire et al., 2007). By analogy, the present results

suggest that the inhibition of Kv7 channels via P2Y1 receptors is the basis for the facilitation of noradrenaline in both, PC12 cells and SCG neurons.

In hippocampal neurons, activation of P2Y1 receptors was found to lead to an inhibition of transmitter release (Rodrigues et al., 2005; Heinrich et al., 2008). Although the underlying

mechanisms remained obscure, endogenous as well as recombinant P2Y1 receptors are known to mediate an inhibition of neuronal voltage-gated Ca2+ channels (Hussl & Boehm, 2006), and this is a prime mechanism of presynaptic inhibition (Brown & Sihra, 2008). 2+ Recently, P2Y1 receptors were also reported to mediate an inhibition of Ca currents in SCG neurons (Filippov et al., 2010). In the preparation used for this study, ADP also elicits an 2+ inhibition of voltage activated Ca channels, but this latter effect excludes P2Y1 and is

mediated only by P2Y12 receptors (Kulick & von Kugelgen, 2002; Lechner et al., 2004). The reasons for this discrepancy remain enigmatic, but it is obvious that an inhibition of voltage- activated Ca2+ channels cannot be the basis for the presynaptic facilitation as described here, but rather underlies presynaptic inhibition.

76 It appears puzzling that one GPCR type like P2Y1 can mediate presynaptic facilitation or presynaptic inhibition depending on the neuron being investigated. However, this has also been found with other presynaptic Gq-linked receptors, for instance with M1 muscarinic acetylcholine receptors: On one hand, M1 receptors mediate an enhancement of noradrenaline release from sympathetic nerve terminals through an activation of protein kinase C (Costa et al., 1993; Somogyi et al., 1996), on the other hand these receptors mediate presynaptic inhibition through the depletion of membrane phosphatidylinositol 4,5- bisphosphate via PLC and the resulting closure of voltage-activated Ca2+ channels (Kubista

et al., 2009). As mentioned above, P2Y1 receptors have been reported to inhibit Kv7 channels as well as voltage-activated Ca2+ channels in SCG neurons, and the latter effect is determined by the presence or absence of a scaffold protein (Filippov et al., 2010). Hence, depending on the scaffold proteins nerve terminals are endowed with, one type of GPCR might mediate presynaptic facilitation or inhibition.

Interactions between two different P2Y receptors It is already established that different types of P2Y receptors, NTPDases and ion channels are expressed at the presynaptic terminal (Lechner et al., 2004) and it would be interesting to

know, if they interact with each other. Thus, the fluorescent versions of P2X2, P2Y1, P2Y12, NTPDase1 and NTPDase2 were studied via FRET microscopy. Ionotropic receptors are known to be composed of several subunits, the formation of homo- and heteromers by GPCRs has been found out (Terrillon & Bouvier, 2004). P2X receptors are known to exist as trimers (Khakh & North, 2006) and P2Y receptors are known to exist as homo- or heterodimers (Koles et al., 2008). Although FRET microscopy has been used to identify the formation of GPCR oligomers for a large number of receptor pairs, the results should be interpreted very cautiously for potential artefacts (Milligan & Bouvier, 2005; Pfleger & Eidne, 2005). Fusion of fluorescent proteins to the above mentioned P2 receptors and

NTPDases might impair their membrane targeting and function, for instance for P2Y1

receptors (Vohringer et al., 2000). Thus the fusion proteins of P2X2, P2Y1, P2Y12, NTPDase1 and NTPDase2 were tested for their functional properties by comparing them to the untagged versions by patch clamp and phosphatase assays, which showed that there is no functional difference between untagged and tagged versions of the proteins (Schicker et al.,

77 2009). Having established that functional fluorescent proteins do reach the membrane, their

targeting was corroborated by confocal laser microscopy for two prototypic examples (P2X2

and NTPDase-1). Using the pairs P2X2-C/P2X2-Y and P2X2-C/NTPD1-Y, positive and

negative controls were established, respectively, for the following reasons: P2X2 has

previously been found to oligomerize in the membrane (Fisher et al., 2004), NFRET values

obtained with P2X2-C/P2X2-Y were identical with those obtained for CFP- and YFP-labeled human SERT and mGluR5, which had also been shown to homooligomerize in cell

membrane, NFRET values obtained with P2X2-C/NTPD1-Y in the membrane were not different from those obtained with only the fluorescent moieties, but the latter were

determined in cytosolic regions and NFRET values for P2X2-C and NTPD1-Y were also

identical with the NFRET values for unrelated pairs of fluorescent membrane proteins that had been established not to interact with each other (Schmid et al., 2001; Calo et al., 2005). The

fluorescent versions of P2Y1, P2Y12 and NTPDase1 were also studied by confocal microscopy for their membrane targeting and colocalisation (data not shown here). FRET microscopy on fluorescent membrane proteins does, rely on the evaluation of recombinant instead of native proteins, and therefore over expression is a frequently mentioned concern, as exaggerated over expression can be expected to force membrane proteins to directly interact with each other. Moreover, over expression might lead to the presence of membrane proteins in membrane compartments in which they would not be located under native conditions. Therefore, reliable positive and negative controls must be established for each of the fluorescent proteins being investigated to certify that the fusion proteins do not lead to apparently positive FRET values by a default mechanism. In table 2,

all the positive interactions as defined by high NFRET values are listed together with all the negative results for all pairs of proteins investigated. A look at this table reveals that none of the fusion proteins employed gave either negative or positive results only. Hence, in no case the presence or absence of interactions with other membrane proteins appeared to be a default mechanism. To further confirm the specificity of positive interactions, a FRET competition assay was performed as frequently recommended for FRET investigations of

GPCRs (Pfleger & Eidne, 2005). In fact, an appropriate unlabelled interaction partner (P2Y1)

reduced the NFRET values obtained with P2Y1-C and P2Y1-Y, whereas a non-interacting

78 protein (NTPDase2) failed to do so. Taken together, all control experiments demonstrate that

the interactions as defined by NFRET values through FRET microscopy are not artefactual.

P2X NTP NTP P2Y P2Y 2 Dase1 Dase2 1 12

P2X2 + - n.a. n.a. n.a.

NTPDase - + n.a. + + 1

NTPDase n.a. n.a. n.a. - n.a. 2 P2Y1 n.a. + - * + * +

P2Y12 n.a n.a. + + +

Table 2 Summary of interactions as defined by NFRET values in comparison with the values

for the positive and negative controls. +, NFRET values not different from those for P2X2-

C/P2X2-Y; −, NFRET values not different from those for P2X2-C/NTPD1-Y; n.a., not analyzed; *, result confirmed by FRET competition. A section of results also taken from (Schicker et al., 2009).

Table 2 reveals that the G-protein coupled P2Y1 and P2Y12 receptors display a high tendency to form homo- and heteroligomeric complexes in the membrane. Homomeric assemblies

have been previously reported for a number of GPCRs including P2Y1 (Choi et al., 2008)

and P2Y12 (Savi et al., 2006). Here both the GPCRs studied for homooligomeric assemblies were found to closely associate in the membrane. Taken together, the GPCRs studied here form not only homooligomers, but also heterooligomers at the membrane.

Thus the results suggest that, P2Y1 and P2Y12 receptors form homo or heterooligomers at the membrane, which upon activation by a common agonist like ADP, exhibit two different

functions at the rat sympathetic nerve terminals namely: Gi-coupled P2Y12 receptors mediate an inhibition of via VGCCs and also mediate an inhibition of NA release from rat SCG

79 neurons (Lechner et al., 2004), whereas Gq-coupled P2Y1 receptors upon stimulation by ADP

mediate a facilitation of noradrenaline release and also mediate an inhibition of IM. Thus in the future, it would be interesting to investigate the interactions between the two P2Y

receptors studied here with the Kv7 and the CaV2.2 channel subunits, which are known to express on the sympathetic neuronal membrane and finding out their functional consequences both in the native and also in the heterologous expression systems. It would also be of interest to dissect out the downstream signaling cascades that might throw some light in understanding the whole mechanism in detail.

80 7 CONCLUSIONS The present results demonstrate that ADP controls sympathetic transmitter release not only

via inhibitory presynaptic P2Y12 (and maybe 13) receptors, but also via facilitatory

presynaptic P2Y1 receptors. The principle that two separate GPCRs for one transmitter family, such as adenine nucleotides, mediate opposing effects at sympathetic nerve terminals is not unknown: noradrenaline and adrenaline activate presynaptic α2 and β2 adrenoceptors and thereby cause inhibition and facilitation of noradrenaline release, respectively (Boehm et al., 2002). Since noradrenaline and adenine nucleotides are cotransmitters in postganglionic

sympathetic neurons, the pair of inhibitory presynaptic P2Y12 and facilitatory presynaptic

P2Y1 receptors can be viewed as a novel counterpart of the well established presynaptic adrenoceptors. The FRET interaction studies revealed that GPCRs such as P2Y can associate either as homomers or heteromers (in more than 10 different heteromeric combinations (Schicker et al., 2009)). Thus, the physiological effects of adenine nucleotides and nucleosides depend on a membrane network of receptors, enzymes and ion channels rather than on the presence of individual proteins.

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104 9 ABBREVIATIONS

AC, adenylyl cyclase; Ach, acetylcholine; ADP, adenosine diphosphate; AIS, axon initial segment; AMP, adenosine monophosphate; AMPA, α-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid; ATP, adenosine triphosphate; AR-C69931MX, N6-(2-methyl- thioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloro-methylene-ATP; BAIRs, the brain specific angiogenesis-inhibitory receptors; BFNC, benign familial neonatal convulsions; CALCR, calcitonin receptor; CASR, calcium-sensing receptor; CCN, cholecystokinin; CNS, central nervous system; CRHRs, corticotropin-releasing hormone receptors; DAG, diacyl glycerol; DA, dopamine; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; Enk, enkephalins; E-NTPDases, ecto-nucleoside triphosphate diphosphohydrolases; EPSP, excitatory postsynaptic potential; FRET, fluorescence resonance energy transfer; FPRs, formyl peptide receptors; FZDRs, frizzled receptors; GABA, Gamma-aminobutyric acid; GCGR, the glucagon receptor; GHRHR, growth hormone-releasing hormone receptor; GIPR, gastric inhibitory polypeptide receptor; GIRK, G-protein-coupled inwardly rectifying K+ channel; GLPRs, glucagon-like peptide receptors; Glu, glutamate; Gly, glycine; GPCR, G protein coupled receptor; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; H-7; + 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; HVA, high-voltage-activated; IM, M-type K

current; IP3, inositol 1,4,5 triphosphate; IPSP, inhibitory postsynaptic potential; KARs, 2+ + + kainate receptors; KCa, Ca -activated K channel; Kir, inward rectifying K channel; LBD, ligand binding domain; LTP, long-term potentiation; LVA, low-voltage-activated; mAChRs, muscarinic acetylcholine receptors; MCHRs, melanin-concentrating hormone receptors; MECARs, melanocortin, endothelial differentiation sphingolipid, cannabinoid and adenosine receptors; mGLUR, metabotropic glutamate receptor; MRS 2179, 2′-Deoxy-N6- methyladenosine 3′,5′-bisphosphate tetrasodium salt; MRS 2365, [[(1R,2R,3S,4R,5S)-4-[6- Amino-2-(methylthio)-9H-purin-9 -yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt; MRS2500, 2-iodo-N6-methyl-(N)- methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate; 2-MeSATP, 2-methylthio-ATP; NA,

noradrenaline; nAChRs, nicotinic acetylcholine receptors; NaV, voltage-gated sodium channel; NCS, neuronal calcium sensor; NMDA, N-methyl-D-aspartate; NTS, nucleus tactus solitarius; PACAP, pituitary adenylyl cyclase-activating protein; PI4K, phosphoinositide 4-

kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein

105 kinase C; PNS, peripheral nervous system; PTHR, parathyroid hormone receptors; PTX, pertussis toxin; RIM, rab3 interacting molecule; SCG, superior cervical ganglion; SCTR, secretin receptor; 5-HT, serotonin; SERT, serotonin transporter; SMOHRs, smoothened; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor, SOGR, suboesophageal ganglion receptor; TASRs, taste receptors; TMD, transmembrane domain; TTX, tetrodotoxin; UDP, uridine di phosphate; UTP, uridine tri phosphate; U73122, 1-[6- [((17β)-3-Methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione; VIP, vasoactive intestinal polypeptide; VIPR, vasoactive intestinal peptide receptor; VGCCs, voltage-gated calcium channels; XE991, 10,10-bis(4-Pyridinylmethyl)-9(10H)-anthracenone dihydrochloride; ZACRs, zinc-activated receptors.

106 10 ACKNOWLEDGEMENTS

Prof. Dr. Stefan Boehm Department of Neurophysiology and -pharmacology Center for Physiology and Pharmacology MedicalUniversity of Vienna Waehringerstrasse 13a A-1090 Vienna Austria.

I would like to extend my sincere thanks to Prof. Klaus Groschner, University of Graz, Austria for his continuous support. I would also like to thank each and every lab member of his lab especially Dr. Michael Poteser, for their wonderful support during my stay.

I would also like to sincerely thank my thesis committee member Prof. Werner Sieghart, Center for Brain Research, Medical University of Vienna for his continuous support.

My sincere thanks also to Mr. T.R. Ganesh and family.

I would like to thank my colleagues and friends for their continuous support both professionally and personally, Klaus W Schicker, Helmut Drobny, Gabi Gaupmann, Simon Hussl, Oliver Kudlacek, Arsalan Yousuf, Isabella Salzer, Mario Dorotksar, Felicia Popovici, Kristina Kosenberger, Petra Geier, Amulya Nidhi Shrivastava, Abhisek Chatterjee, Adhar Talati, Ashwin Kumar Kayoor, Dhairyasheel Desai, Harsh Patel, Juhi Sinha, Manoj Mishra, Mihir Gajjar, Nischita Mistry, Nisha Geetha, Rakhi Sharma, Revu Ann Alexander, Shaurya Anand, Sravan Payeli, Subhodeep Sarker, Suman Vangala, Syam Yelamanchi, Vikash Kumar, many more to name especially friends and colleagues from CCHD graduate school and the Department of Pharmacology, MUV. With due respect, I also would like to sincerely thank all my teachers since childhood, who have taught not only the basics in academia but also have played an important role in moulding my character.

Financial support by CCHD graduate school funded by FWF and the Medical University of Vienna is gratefully acknowledged.

107 11 CURRICULUM VITAE

GIRI KUMAR CHANDAKA

Personal Data

Date of birth: May 04, 1980

Place of birth: Visakhapatnam, India

Nationality: Indian

Email: [email protected], [email protected]

Mobile number: 00436505609161

Education

1985 – 1995: Schooling, Kendriya Vidyalaya No. II, 104 area, Visakhapatnam, India.

1995 – 1997: Intermediate, Visakha Junior College, Dwarakanagar, Visakhapatnam, India.

1998 – 2001: B.Sc in Zoology, Biochemistry and Human Genetics, M.P.R. Degree College, Gajuwaka, Visakhapatnam, India.

2001 – 2003: M.Sc in Human Genetics, Andhra University, Visakhapatnam, India.

2004 – 2006: M.S in Molecular Biology, Umea University, Umea, Sweden.

2007 – Present: pursuing PhD, through an International Graduate Programme (CCHD), at the Department of Pharmacology, Medical University of Vienna, Vienna, Austria. (Degree expected in September 2011)

108 Career history

2003 – 2004: Chromosomal abnormalities in spontaneous aborters. Department of Human genetics, Andhra University, Visakhapatnam, India.

2004 (Oct) – 2005 (Jan): Structural basis of amyloid formation studied by Atomic Force Microscopy (AFM) and spectroscopic techniques, implications to cellular toxicity. Department of Medical Biochemistry and Biophysics, Umea University, Umea, Sweden.

2005 (Jan) – 2005 (June): Examining Fak56 function in the Drosophila nervous system. Department of Molecular Biology, Umea University, Umea, Sweden.

2005 (Oct) – 2006 (Jan): Phenotypic and molecular description of selected Arabidopsis thaliana SALK lines. Department of Plant Physiology (UPSC), Umea University, Umea, Sweden.

2006 (Feb) – 2006 (April): Genetics of retinal degenerations in northern Sweden. Department of Medical and Clinical Genetics, Umea University, Umea, Sweden.

2006 (May) – 2006 (July): Short term training course for neuroscience research at the University of Saarland, Germany.

2009 (July) – 2009 (December): Guest scientist/researcher, in the Department of Pharmacology, University of Graz, Graz, Austria.

2007 – Present: pursuing PhD, through an International Graduate Program, at the Department of Pharmacology, Medical University of Vienna, Austria.

Teaching Experience

Taught a course in 2009 for the PhD students in the Medical University of Vienna: “Fluorescent Resonance Energy Transfer (FRET) as a tool to study Protein Interactions”.

109 Awards

2009: Federation of European Neuroscience Societies (FENS) and Polish Neuroscience Society (PNS) Fellowship for presenting my data in Warsaw, Poland.

Publications

Chromosomal aberrations in recurrent aborters. Giri Kumar Chandaka, Sunil Kumar P, Lakshmi Kalpana, Satyanarayana M. Bionature 2004.

A membrane network of receptors and enzymes for adenine nucleotides and nucleosides. Klaus Schicker, Simon Hussl, Giri K. Chandaka, Kristina Kosenburger, Jae-Won Yang, Maria Waldhoer, Harald H Sitte, Stefan Boehm. Biochim Biophys Acta. 2009.

+ P2Y1 receptors mediate an activation of neuronal calcium-dependent K channels. Klaus Schicker, Giri K. Chandaka, Petra Geier, Helmut Kubista, and Stefan Boehm. The Journal of Physiology. 2010.

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y1 receptors. Giri K. Chandaka, Isabella Salzer, Klaus W. Schicker, Helmut Drobny, and Stefan Boehm. British Journal of Pharmacology. 2011.

Few more papers are in pipeline.

Abstracts for Conferences

International Workshop On Genetics Of Complex Diseases With an Emphasis On Type 2 Diabetes Mellitus” organized by Andhra University and Andhra Medical College, Visakhapatnam, India in collaboration with Southwest Foundation for Biomedical Research and University of Texas Health Science Centre, San Antonio, Texas, USA, in 2003.

PhD Symposium of the Young Scientists Association (YSA) at the Medical University of Vienna, Vienna, Austria, June 2007.

Presynaptic P2Y1 receptors in Sympathetic Neurons. Giri K. Chandaka, Helmut Drobny, Stefan Boehm. Research Retreat of the Center for Biomolecular Medicine, Physiology and Pharmacology, MUV, Vienna, Austria. February 29th, 2008.

Presynaptic P2Y1 receptors in sympathetic neurons. Giri K. Chandaka, Helmut Drobny, Stefan Boehm. PhD Symposium of the Young Scientists Association (YSA) at the Medical University of Vienna, Vienna, Austria, May 28th - 29th, 2008.

110

Facilitation of transmitter release from rat Sympathetic neurons via presynaptic P2Y1 receptors. Giri K. Chandaka, Helmut Drobny and Stefan Boehm. Research Retreat of the Center for Biomolecular Medicine, Physiology and Pharmacology, MUV, in Sopron, Hungary, September 5th – 6th, 2008.

A membrane network of receptors and enzymes for adenine nucleotides and nucleosides. Klaus Schicker , Simon Hussl, Giri K. Chandaka, Kristina Kosenburger, Jae-Won Yang, Maria Waldhoer, Harald H Sitte, Stefan Boehm. APHAR (Austrian Pharmacological Society) meeting, in Innsbruck, Austria, November 21st - 22nd, 2008. BMC Pharmacology. Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y1 receptors. Giri K. Chandaka, Helmut Drobny and Stefan Boehm. APHAR (Austrian Pharmacological Society) meeting, in Innsbruck, Austria, November 21st - 22nd, 2008. BMC Pharmacology.

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y1 receptors. Giri Kumar Chandaka, Helmut Drobny and Stefan Boehm. Society for Neuroscience (SFN) meeting in Washington DC, USA, November 15th – 19th, 2008.

Interplay between P2Y receptors and KV7 K+ channels. Giri Kumar Chandaka & Stefan Boehm. Research Retreat of the Center for Biomolecular Medicine, Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria. April 17th – 18th, 2009.

Actions of P2Y1 receptors in sympathetic neurons. Giri Kumar Chandaka, Helmut Drobny and Stefan Boehm. PhD Symposium of the Young Scientists Association (YSA) at the Medical University of Vienna, Vienna, Austria, June 17th - 19th, 2009.

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y1 receptors. Giri K. Chandaka, Helmut Drobny, and Stefan Boehm. Polish Neuroscience Society (PNS) and Federation of European Neuroscience Societies (FENS) meeting in Warsaw, Poland, September 09th – 12th, 2009. Acta Neurobiologiae Experimentalis, Volume 69, No. 3 (pp: 263-377).

Functional interactions between P2Y receptors and M-type K+ channels. Giri Kumar Chandaka, Klaus Groschner and Stefan Boehm. PhD Symposium of the Young Scientists Association (YSA) at the Medical University of Vienna, Vienna, Austria, June 16th - 17th, 2010.

Functional interactions between P2Y receptors and M-type K+ channels. Giri Kumar Chandaka, Klaus Groschner and Stefan Boehm. Research Retreat of the Center for Biomolecular Medicine, Physiology and Pharmacology, Medical University of Vienna in Sopron, Hungary, September 10th – 11th, 2010.

111 Extracurricular Activities

Elected student deputy speaker of the CCHD international graduate program between 2007 and 2010, in the medical university of Vienna, Vienna, Austria.

Student representative during my bachelor’s degree, in M.P.R. degree college, Visakhapatnam, India.

Chief organiser for various academic and social meetings/gatherings.

112