Department of Neurology and Neurophysiology Medizinische Hochschule Hannover
and
Centre for Systems Neuroscience Hannover University of Veterinary Medicine Hannover
Pharmacokinetic properties of selective and non-selective AMPA-receptor antagonists and their role in neuroprotection
Thesis
Submitted in partial fulfilment of the requirements for the degree
-Ph.D.-
In the field of Neuroscience
At the Centre for Systems Neuroscience Hannover Awarded by the University of Veterinary Medicine Hannover
by
Dr. med. Friedrich Schlesinger Falkenberg
Hannover, 2007
Supervisor: Prof. Dr. med. J. Bufler and PD Dr. med. K. Krampfl
Scientific advisors: Prof. Dr. med. R. Dengler and Prof. Dr. rer. nat. H-A.Kolb
External examiner: Prof. Dr. med. M. Heckmann
Date of final exam: 02.11.2007
Publications:
1 Schlesinger F, Tammena D, Krampfl K, Bufler J. Desensitization and resensitization are independently regulated in human recombinant GluR subunit coassemblies. Synapse. 2005 Mar 1;55(3):176-82.
2 Schlesinger F, Tammena D, Krampfl K, Bufler J. Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors. Br J Pharmacol. 2005 Jul;145(5):656-63.
3 Krampfl K, Schlesinger F, Cordes AL, Bufler J. Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels. Neuropharmacology. 2006 Mar;50(4):479-90.
Index
1 Glutamate receptors 4
1.1 Electrophysiological characteristics of AMPA receptors 7
2. Chronic excitotoxicity in Amyotrophic Lateral Sklerosis (ALS) and neuroprotection 8
3. Materials and methods 10
3.1 Solutions, Cell culture and transfection 10
4 Patch-clamp technique 11
4.1 Equipment 11
4.2 Patch-Clamp Technique 13
4.3 Fast application technique 16
5 Disscusion 17
6 Summary 21
6 Zusammenfassung 23
7 Reference list 26
Publications
Desensitization and resensitization are independently regulated in human recombinant GluR subunit coassemblies. Schlesinger et al. 2005
Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA- type glutamate receptors. Schlesinger et al. 2005
Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels. Krampfl et al.
2006
Acknowlegement
Introduction
1. Glutamate receptors
Glutamate receptors can be divided into two groups, the G-protein second messenger system dependent and mediated metabotropic glutamate receptors (mGluRs) and the ionotropic ligand-gated receptors (Simeone et al., 2004). The ionotropic receptors can further be subdivided into three classes according to their preferred synthetic agonist (Hollmann & Heinemann, 1994): AMPA (α-amino-3- - hydroxy-5-methyl-4-isoxazolepropionic acid), NMDA (N-methyl-D-aspartate) and kainate receptors.
NMDA receptors mediate the late component of excitatory transmission (Collingridge
& Lester, 1989) and play a key role in the induction of synaptic plasticity (Nicoll &
Malenka, 1999) an acute excitotoxicity (von Engelhardt et al., 2007)
The role of kainate receptors in physiological and pathological conditions is not clear so far.
Among those glutamate receptors, AMPA receptors play a central role in fast excitatory transmission. It was described by different authors that motor neurons in culture are particularly susceptible to glutamate receptor agonists, especially to AMPA receptor agonists (Van Den Bosch & Robberecht, 2000). AMPA receptors are permeable to Na+, K+, and some for Ca2+. Ca2+ influx through Ca2+-permeable AMPA receptors is crucial for triggering motor neuron death (Van Den Bosch et al., 2000
&2002). Intrathecal or intraspinal administration of AMPA receptor agonists has been shown to induce motor neuron degeneration in animals, whereas NMDA failed to damage spinal motor neurons (Kruman et al., 1999; Hugon et al., 1989;
Ikonomidou et al., 1996). In organotypic rat spinal cord cultures, motor neurons were also proved to be vulnerable to AMPA receptor-mediated excitotoxicity (Saroff et al., 2000; Rothstein et al., 1993). Direct application of AMPA receptor agonists resulted in selective motor neuron loss, which could be prevented by antagonists of AMPA receptors. For that reason, we focus on AMPA-type receptors when investigating the pharmacological interactions of several candidate compounds for clinical neuroprotection.
AMPA-type Glutamate receptor channels show a variable assembly of four individual subunits (GluR1–4) (Wu et al., 1996; Mano & Teichberg, 1998; Safferling et al., 2001;
Keinanen et al., 1990, Rosenmund et al., 1998). The schematic topology (Palmer et al., 2005) of an AMPA-type receptor subunit is illustrated in Fig.1.1. The molecular architecture of each AMPA-type receptor subunit is very similar; each comprises
~900 amino acids and has a molecular weight of ~105 kDa37. There is approximately 70% sequence homology between genes encoding each subunit, although genes may undergo alternative splicing in two distinct regions, resulting in subunits that have either long or short C termini, and flip or flop variants in an extracellular domain (Palmer et al., 2005).
Fig.1.1 Model of an AMPA receptor subunit. Each subunit consists of an extracellular N-terminal domain, four hydrophobic regions (TM1–4), and an intracellular C-terminal domain. The ligand-binding site is a conserved amino acid pocket formed from a conformational association between the N terminus and the loop linking TM3 and TM4. A flip/flop region (alternative splicing) and R/G RNA editing site are also present within the TM3/TM4 loop. TM2 forms an intracellular re-entrant hairpin loop which contributes to the cation pore channel and is also the site for Q/R RNA editing in the GluR2 subunit.
Each AMPAR-subunit includes a channel-forming domain consisting of three transmembrane domains, TM1, TM3, and TM4 and a re-entrant loop TM2, an intracellular C terminus, and two extracellular domains composed of the N terminus and the segment between TM3 and TM4 (S2) (Fig.1.1). The N terminus contains the
N-terminal domain of ~400 amino acids and a ~150 amino acid ligand-binding segment (S1), which forms an extracellular agonist-binding domain with S2.
The intracellular C terminus of eukaryotic AMPA-type receptor channels has been shown to be the site of interaction for a range of different proteins, many of which are involved in the receptor trafficking (Henley, 2003) and synaptic plasticity (Malenka, 2003).
Each of the four AMPA receptor subunits exists at least in two splice variants, named flip and flop (Sommer et al., 1990) (see Fig.1.1), generated by alternative splicing of two 115-base pair exons that encode 38 amino acids located in the extracellular loop between TM3 and TM4. This modifies the channel’s kinetic and pharmacological properties. The receptors with flip splice variants desensitize four times slower than that with flop (Mosbacher et al., 1994; Koike et al., 2000) and the flip module confers different sensitivity to allosteric modulators cyclothiazide (Partin et al., 1994; Kessler et al., 2000), 4-[2-(phenylsulfonylamino) ethylthio]-2,6 -difluoro- phenoxyacetamide (Sekiguchi et al., 1998), zinc (Shen & Yang, 1999), and lithium (Karkanias & Papke, 1999), although affinity to AMPA is unchanged (Arvola & Keinanen, 1996).
The genomic DNA of the GluR2 subunit of the AMPA-type receptor contains a glutamine (Q) residue at amino acid position 607 (Q/R site). However, the majority of neuronal mRNA contains a positive charged arginine (R) at this position due to a process of nuclear RNA editing (Sommer et al., 1991). Receptors containing edited GluR2 have a very low Ca2+ permeability (Hollmann et al., 1991), low single-channel conductance (because of the size and charge of the amino acid side chain in the edited form) (Burnashev et al., 1996; Swanson & Kamboj, 1997). Nonetheless, GluR2 (R)-containing AMPA-type receptor channels can still participate in intracellular Ca2+ signalling (Utz & Verdoorn, 1997) and can be trafficked in a Ca2+- dependent way (Liu & Cull-Candy, 2000).
The R/G editing site is located near to the flip/flop splice region (Lomeli et al., 1994)(Fig.1.1). This modification changes the desensitization and resensitization of the resulting AMPA-type receptor channels (Krampfl et al., 2001).
The following established nomenclature is used:
Glu(tamate) R(ezeptor)1-4 i/o(flip/flop) R(Glutamin)/G(Glycin) and Q(Arginin/R(Glutamin)
e.g. GluR2iGQ
Fig.1.2 The Ca2+ permeability of AMPA receptors is determined by the presence or absence of edited GluR2 in the receptor complex. AMPA receptors composed of a combination of GluR1-3-4 are permeable to Ca2+ ions (left). A positively charged arginine at position 607 in GluR2 prevents the permeation of Ca2+ ions, when present in the receptor complex (right).
1.1 Electrophysiological characteristics of AMPA receptors
Figure 1.3 shows an example for the current response of GluR2 flip-receptors upon application of glutamate at a saturating concentration recorded with the Patch- Clamp-Technique. After fast application of glutamate the current transient develops with a fast rise time (TTP) to the maximum peak current amplitude. In prolonged presence of the agonist the current decreases to a „steady-state“ current amplitude due to desensitization. After removing the agonist, the current reduced continuously to zero level due to deactivation.
Fig.1.3 The electrophysiological characteristics of AMPA receptor currents
2. Chronic excitotoxicity in Amyotrophic Lateral Sklerosis (ALS) and neuroprotection
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord, which results in progressive paresis of bulbar, respiratory and limb muscles, while sensory and cognitive functions are preserved in most cases. ALS typically affects adults in mid-life, with an incidence of 1–2/100,000/year (Van Damme et al., 2005). Neurological examination reveals lower motor neuron signs and upper motor neuron signs. The progressive muscle weakness results in death after 2–5 years following the onset of the disease, mostly due to respiratory failure. Treatment remains essentially supportive, since no substantial therapy is available. Riluzole, the only drug proven to slow down disease progression, prolongs survival of ALS patients by a few months
(Bensimon et al., 1994, Lacomblez et al., 1996)
The pathogenic mechanism of the selective motor neuron death in ALS is not known so far. In 5–10% of cases familial ALS occurs, predominantly showing an autosomal
dominant inheritance. In 20% of familial cases (which represents only 1–2% of all cases), mutations in the superoxide dismutase-1 (SOD1) gene on chromosome 21q were identified (Rosen et al., 1993). Mutations in alsin (Yang et al., 2001), in senataxin (Chen et al., 2004), or in the gene encoding the vesicle-trafficking protein (VAPB) (Nishimura et al., 2004) were also reported. But the majority of ALS patients has no affected family members and is considered to have sporadic ALS. A number of possible mechanisms have been proposed (Cleveland & Rothstein, 2001, Brown &
Robberecht, 2001, Heath & Shaw, 2001, Julien, 2001). These include oxidative stress, axonal strangulation from neurofilamentous accumulations, toxicity from intracellular protein aggregates, mitochondrial dysfunction, inflamation, decreased availability of growth factors and excitotoxic death of motor neurons.
Excitotoxicity is undoubtedly one of the most robust pathogenic mechanisms, which is neuronal degeneration induced by overstimulation of glutamate receptors and can occur when the extracellular glutamate concentration increases or when the postsynaptic neuron becomes vulnerable to normal glutamate levels. The amino acid glutamate is the major excitatory neurotransmitter in the mammalian central nervous system with low extracellular glutamate concentration (approximately 0.6 µM) (Benveniste et al., 1984), in spite of a high intracellular concentration of approximately 10 mM (Kvamme et al., 1985) and frequent release of glutamate at glutamatergic synapses. Elevation of the extracellular glutamate concentration to 2–5 µM is considered sufficient to cause degeneration of neurons through excessive stimulation of glutamate receptors (Meldrum & Garthwaite, 1990, Rosenberg et al., 1992). This can occur when the release from presynaptic terminals is augmented or when the re-uptake from the synaptic cleft is insufficient. In addition, lethal injury to neurons, astrocytes or microglia can lead to the release of the intracellular glutamate content. At the postreceptor level, energy depletion renders neurons more vulnerable to excitotoxicity, resulting in neuronal damage in the presence of normal glutamate levels (Novelli et al., 1988, Henneberg et al., 1989).
Motor neurons are extremely sensitive to excitotoxicity, as they have not only a limited capacity to buffer Ca2+ rises due to the low amount of Ca2+-buffering proteins in their cytosol, but also have a high proportion of Ca2+-permeable AMPA-type glutamate receptors and stimulation of these receptors leads to selective motor neuron death (Carriedo et al., 1996, Van Den Bosch et al., 2000 & 2002).
Antagonizing glutamatergic neurotransmission by blockade of AMPA type glutamate
receptors is a promising pharmacological strategy for neuroprotection in neurodegenerative diseases, like ALS.
3. Materials and methods
3.1 Solutions, Cell culture and transfection
Transformed human embryonic kidney (HEK)293 cells were cultured in culture medium at 37°C in a 5% CO2/95% air incubator. The HEK 293 cells were cultured before and after transfection in Dulbeccos modified Eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 g/ml streptomycin.
The electroporation buffer for electric transfection contained: 50mM K2HPO4, 20 mM K-acetate, pH 7.35.
The extracellular solution for HEK293 cells, with which were the cells continuously perfused during each measurement contained 162 mM NaCl, 5.3 mM KCl, 2 mM CaCl2, 0.67 mM NaHPO4, 0.22 mM KH2PO4, 15 mM Hepes, 5.6 mM glucose. The pH value was adjusted with NaOH to 7.4. The solution was filtered each time before use.
Patch pipettes were filled with intracellular solution: 140 mM KCL, 11 mM EGTA, 10 mM Hepes, 10 mM glucose, 2 mM MgCl2. The osmolarity was adjusted to 340 mosM/l with mannitol. The pH value was adjusted with KOH to 7.3.
Chemicals (e.g. IEM-1460) were purchased from Sigma (USA) except sodium-L- glutamate (monohydrate) which was obtained from Merck (USA) RPR119990 and RPR117824 which were kindly provided by Sanofi-Aventis, Paris, France.
Glutamate, RPR119990, RPR117824, IEM -1460 have good solubility in water.
Before each experiment the tested substances were freshly solved in extracellular solution at different concentration yielding a clear solution.
Brillant green was used as a dye for better visualisation of the liquid filament of test solution within the recording chamber.
For transfection, cells were resuspended in electroporation buffer to a density of approximately 8×105 cells/ml. MgSO4 was added to a final concentration of 25 mM.
0.4 ml of this cell suspension was added to purified plasmid DNA (10 µg for each GluR subunit), green fluorescent protein (GFP) 5 µl and mixed. Electroporation at room temperature was performed instantly after the addition of cells to DNA in 0.4- cm cuvette at 250V, 750µF and 329 Ω, resulting in a pulse time of 18-21 ms.
Transfected cells were plated on 12-mm glass coverslips and incubated for 15-24 h prior to the experiments. The transfected cells express glutamate receptors and GFP, which can be visualized using fluorescence microscopy.
4 Patch-clamp technique
4.1 Equipment
Patch-clamp experiments were performed on a vibration isolated table (low-end air- suspension table), to produce mechanical stability and avoid unintended relative movements of the pipette/cell. In the center of the table an upright microscope is placed (Zeiss, amplification up to 40x, water-immersion-objective, fluorescence equipped), which can be moved vertically to allow focusing. The stage of the microscope is fixed on a small air-suspension table which reduces vibrations.
The preamplifier is included in the pipette-holder moved by a hydraulic system. The micromanipulator is connected to the pipette-holder.
Fig.2.1 The patch-clamp setup: (1) Faraday cage, (2) vibration isolation table, (3) microscope, (4) micromanipulator with preamplifier and pipette-holder, (5) stage with bath-perfusion-system and fast-application-system. (6), control of the micromanipulator, (7) first outlet of fast perfusion system, (8) second outlet of perfusion system, (9) hydraulic pump for the drainage and overflow of bath solution, (10) Analog-Digital-converter, (11) video camera, (12) power supply for lamp of microscope (13) oscilloscope, (14) patch-clamp amplifier, (15) computer for signal recording and analysis.
In the center of the stage there is the chamber with perfusion- and drainage- systems, in which coverslips with cells can be perfused with extracellular solution.
The test solutions will be applied via a small capillary connected the piezo-driven fast-application-system, located next to the chamber.
4.2 Patch-Clamp Technique
The patch-clamp technique is an electrophysiological method that allows the recording of macroscopic whole-cell or microscopic single-channel currents flowing across biological membranes through ion channels. The technique allows one to experimentally control and manipulate the membrane potential of membrane patches or the whole-cell (voltage clamp). Alternatively, one may monitor the changes in membrane potential in response to currents flowing across ion channels (current clamp), which constitute the physiological response of a cell. Thus, it is widely used to investigate ion channels in excitable membrane preparations.
.
The voltage clamp technique was used in this experimental study. Upon the delivery of a voltage step to an excitable membrane, the elicited current is dependent on the resistance of the pipette – cell network. Stimulation of membrane resident ion channels will increase their open probability and thus increase membrane conductance. The resulting current can be monitored at a constant holding (= clamp) potential. Fig.2.2 shows the final current-to-voltage convert circuit.
Fig.2.2 Principle of Patch-Clamp technique. Vpip: Pipette Voltage = Membrane Voltage in mV; Vref: Reference Voltage= Command Voltage in mV; Vout: Output Voltage in mV; Rf = feedback resistance in Ohm.
The compensation current I is the indirect measured value, which results from the difference of membrane-voltage (Vpip) and command voltage (Vref). The preamplifier (operational amplifier and feedback resistence Rf) injects a proportional current into the pipette, which results from the difference of Vpip and Vref measured in the operational amplifier.
The voltage, resulting from the difference at Rf and the Vref , define the output voltage Vout. Because of “Ohm’s-law” E(V)=R(Ω) x I(A) the output voltage Vout can be given as the compensation current I in A.
Patch pipettes were made from borosilicate glass, which is in the form of standard microelectrode capillaries (HARVARD apparatus LTD, GC150TF-10, 1.5 mm O.D.X 1.17 mm I.D.). The pulling is in two stages using a horizontal microelectrode puller (DMZ – Pipettenpuller, Zeitz-Instrumente, Augsburg) and then heat polishing is done
at the pipette tip. Pipettes are freshly made before each measurement, filled with intracellular solution, and then mounted on a pipette holder. The resistance values of pipettes are in the range of 5 – 12 MΩ. The holder serves two basic functions, firstly to provide electrical connection between the patch pipette solution and a BNC connector of the amplifier head stage which is mounted on a Narishige MO-103 hydraulic micromanipulator, and secondly to allow suction or pressure to be applied to the pipette interior.
The basic approach to measure small ionic currents in the picoampere range requires a low-noise recording technique. This is achieved by sealing patch-pipette tightly onto the plasma membrane of an intact cell, thereby isolating a small patch.
When the tip of the pipette was pressed against the cell surface the seal resistance increased to 20-30 MΩ. The resistance increased to gigaseal-levels when applying a negative pressure. The development of giga-seals usually occurs within several seconds. The seal resistance should be in excess of 109 Ω. This “cell attached”
configuration is the precursor to all other variants of the patch-clamp technique (fig 2.3).
Fig.2.3. Schematic representation of the procedures which lead to recording configurations. The two recording configurations, described in this paper are: " whole- cell model" and "outside-out patch" (modified from Hamill et al. 1981).
4.3 Fast application technique
Outside-out patches or whole-cell patches were moved in a tube where the cell or patch was continuously perfused by the background flow of the double-barrelled application system “Theta-glass system”. This continuous perfusion guarantied the testing of receptor-blocker interaction before application of short pulses of the agonist (preincubationexperiment). Short pulses of agonist or agonist and blocker were applied via the foreground-flow (fig. 2.3.).
The patch was located at one side of the interface for background perfusion. Then, controlled by the recording PC, the interface moved toward the patch pipette, and the test solution was applied to the patch.
A B
Both of the two channels are separately connected with a rotary dial selector, consisting of two barrels with the same high pressure, in which different solutions can be changed. The time necessary for complete exchange between two perfusing solutions at the outlet of the application capillary is about 10~20 sec.
Fig.2.4 Theta-glass application system. Patch-pipette is placed in the background- flow. During short application pulses the Theta-glass moves and the Patch-pipette is placed within the test-solution-flow (foreground-flow).
5 Disscusion
AMPA-type glutamate receptor (GluR) channels provide fast excitatory synaptic transmission within the central nervous system. They are characterized by their fast kinetics of activation, desensitization, deactivation and resensitization, which determine the time course and limit the frequency of excitatory postsynaptic currents (Jones & Westbrook, 1996). The kinetics and biophysical properties of homomeric AMPA-type GluR channels were analyzed in several studies and it was shown that four subunits build up a functional tetrameric receptor (Mansour et al., 2001;
Rosenmund et al., 1998). In contrast to other ligand gated channels such as skeletal muscle nicotinic acetylcholine receptor channels, which build up functional channels in fixed subunit assemblies (Unwin, 1989), GluR channels assemble to functional homomeric as well as heteromeric channels. The occurrence of posttranscriptional editing sites and flip/flop splice variants increases the number of possible receptor
subtypes further. The Q/R/N posttranscriptional editing site of the GluR2 subunit is involved in the regulation of the Ca2+ conductance and rectification properties (Bowie
& Mayer, 1995; Burnashev et al., 1992; Hume et al., 1991; Kamboj et al., 1995; Koh et al., 1995; Lomeli et al., 1994; Seeburg, 1996; Sommer et al., 1991), trafficking processes and subunit assembly (Greger et al., 2003).
In vivo most GluR channels occur as a coassembly of the subunits GluR2 and GluR1, (3 or 4). There is experimental evidence that two subunits coassemble first to dimers and two dimers in a second step to tetrameric receptors (Rosenmund et al., 1998). The idea of a preferential arrangement of subunits (Ayalon & Stern-Bach, 2001) (Brorson et al., 2004; Mansour et al., 2001) arised from the observation that values of specific parameters of homomeric channels are passed on heteromeric channels. In the study “Desensitization and resensitization are independently regulated in human recombinant GluR subunit coassemblies” , Schlesinger et al.
2005, we focused on desensitisation and resensitisation kinetics of different heteromeric GluR channels. Coexpression of GluR2 flop RN and GluR2 flop GN channels expressed in a 2:2 plasmid cDNA ratio resulted in values for the desensitisation (τD) and resensitisation (τrec) just between that of the respective homomeric channels (Krampfl et al., 2002). In contrast, when plasmids of GluR1 flip and GluR2 flip RN channels were cotransfected, we found a clear dependence of τD
on the amount of the transfected cDNA, whereas τrec of these heteromeric channels was as fast as that of homomeric GluR flip R channels (Grosskreutz et al., 2003). To get reliable results, cotransfection of GluR subunit cDNA was performed with subunits, which differed for the values of τD or τrec by at least a factor of two. A predominance of the fast resensitising over slow resensitising channels was shown when cDNA of GluR2 flip channels was cotransfected with that of GluR1 channels, referring to a preferential assembly. However an inverse correlation between τrec and the amount of the specific cDNA plasmids was found exclusively when GluR2 flip was cotransfected with GluR1 channels and in contrast to τrec, τD had intermediate values at all combinations of heteromeric and homomeric channels and was directly dependent on the amount of transfected cDNA. It is concluded from our data that desensitisation and resensitisation kinetics are regulated independently and are dependent on the specific subunit composition of the receptor. The control of the resensitisation of GluR channels in heteromeric receptor assemblies is a so far unknown property of the flip variant of the GluR2 subunit.
Despite there is a broad variety of drugs that modulate and block AMPA-type glutamate receptors in vitro, none of the AMPA- blockers that underwent clinical testing so far were appropriate for a therapeutical use in patients (Doble, 1999). In our study “Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors”, Schlesinger et al. 2005 we tested the recently described glutamate receptor antagonist IEM-1460 for receptor interactions at the molecular level. IEM-1460 binds to Ca2+-permeable AMPA-type glutamate receptor channels and not to Ca2+-impermeable channels (Magazanik et al., 1997;
Tikhonov et al., 2000). This means that currents through AMPA-type channels are blocked by IEM-1460 except the respective channels contain a Q/R edited GluR2 subunit which renders the respective channels Ca2+-impermeable. The IC50 of IEM- 1460, measured at oocytes transfected with GluR1 or 3 subunits after application of 0.1 mM kainate, was ~2 μM (Magazanik et al., 1997).
The sensitivity of human GluR2 flip GN channels to IEM-1460 was the same as that to human GluR2 flip GQ channels, GluR1 channels were double as effectively blocked than GluR2 flip GQ or GluR2 flip GN channels and the mutant GluR2 L504Y channels were most sensitive to IEM-1460 with an IC50 of 15 μM. As was suggested in previous studies (Magazanik et al., 1997; Buldakova et al., 1999), IEM-1460 was nearly ineffective at GluR2 flip GR channels which have a very low Ca2+-permeability.
It was shown that the IC50 is shifted at least by a factor of 1000 to the right when homomeric Ca2+-impermeable GluR2 flip GR channels were tested. This specific effect of IEM-1460 was clearly confirmed by the results of our study and it holds also true when low amounts of cDNA of GluR2 flip GR subunits are used for coexpression at HEK293 cells. The IC50 of native human AMPA-type channels (except GluR2 flip GR channels) was in the range of low sensitivity rat hippocampal neurons (Magazanik et al., 1997). The affinity of IEM-1460 to mutant GluR2 L504Y channels was much higher. Beside the different experimental design of the studies, species differences might play a role for the different affinity of IEM-1460.
In an additional study “Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels”, Krampfl et al. 2006, the two novel neuroprotective AMPA antagonists RPR119990 and RPR117824 were tested for their receptor interactions at the molecular level. Both compounds have neuroprotective activity in different functional assays (Canton et al., 2001; Mignani et al., 2002). Preliminary pharmacological
characterization revealed that both compounds block kainate induced currents at AMPA type glutamate receptors in the nanomolar concentration range (Canton et al., 2001; Mignani et al. 2002). The objective of our patch-clamp analysis was 1) to profoundly investigate the molecular mechanism of action of the compounds, 2) to test for their AMPA-receptor subtype specificity and for their effects on kainate receptor channels. The data should serve as a database for further laboratory and therapeutic use of these highly potent AMPA blockers.
Substitution of the phosphonate group in position 2 by a carboxylic group (see synthesis reports of RPR119990 and RPR117824 in Canton et al., 2001; Mignani et al., 2002) results in a significantly (GluR2 flop; GluR1 flop and GluR2 flip, coexpressed; GluR6) greater blocking activity of RPR117824 compared to RPR119990 at recombinant and synaptic AMPA-receptors. Despite that quantitative differences, the effects of RPR119990 and RPR117824 are discussed together, since we found no substantial difference in the molecular mechanism of AMPA- receptor antagonism. In terms of potency the novel AMPA antagonists overwhelms competitive blockers like CNQX, NBQX, and YM90K (Dingledine et al., 1999, Doble, 1999, Paternain et al., 1996, Turski et al., 1998). Like CNQX, the classical competitive AMPA antagonist (Honoré et al., 1988), RPR119990 and RPR117824 block also kainate receptors by a competitive block mechanism, but with much less efficacy as shown by the IC50 of 2.0 μM for RPR119990 and 480 nM for RPR117824.
The parallel rightward shift of the dose response curve of kainate in the presence of RPR119990 and RPR117824 that was shown by Canton et al. (2001) and Mignani et al. (2002) corresponds well with the data shown in fig. 1 and table 1. Thus, the experimental results of our study add further evidence for the assumption of a competitive-like block mechanism.
For comparison of the quantitative results, we investigated not only homomeric recombinant GluR channels. Because both the GluR1 flop and GluR2 flip subunits show strong coexpression in many regions of the central nervous system, heteromeric GluR1 flop/GluR2 flip channels were expressed in HEK293 cells.
Quantitative analysis revealed well matching data for the channel blocking activity of RPR119990 and RPR117824 in heteromeric as well as homomeric AMPA receptor channels.
In conclusion, we could show that RPR119990 and RPR117824 block AMPA receptors competitively with an outstanding block activity as indicated by an IC50 in
the low nanomolar range. The results hold true for different recombinant GluR channel subtypes. Especially the use of a non-desensitizing recombinant GluR channel enabled us to determine block mechanism and kinetics by co-application protocols. This methodological approach may allow for convenient screening of putative AMPA receptor antagonists and modulators. An IC50 value in the low nanomolar range for AMPA receptor channels in combination with a reduced potency on kainate receptors makes these compounds extremely interesting for a therapeutic use, as relevant blood concentration might be achieved and future studies should settle the issue of AMPA-antagonism in clinical neuropharmacology.
6 Summary
In the study “Desensitization and resensitization are independently regulated in human recombinant GluR subunit coassemblies” , Schlesinger et al. 2005, we focused on desensitisation and resensitisation kinetics of different heteromeric GluR channels. A predominance of the fast resensitising over slow resensitising channels was shown when cDNA of GluR2 flip channels was cotransfected with that of GluR1 channels, referring to a preferential assembly. In contrast, when plasmids of GluR1 flip and GluR2 flip channels were cotransfected, we found a clear linear dependence of τD on the amount of the transfected cDNA.
However an inverse correlation between τrec and the amount of the specific cDNA plasmids was found when GluR2 flip was cotransfected with GluR1 channels and in contrast to τrec, τD had intermediate values at all combinations of heteromeric and homomeric channels and was directly dependent on the amount of transfected cDNA. It is concluded from our data that desensitisation and resensitisation kinetics are regulated independently and are dependent on the specific subunit composition of the receptor.
In our study “Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors”, Schlesinger et al. 2005 we tested the recently described glutamate receptor antagonist IEM-1460 for receptor interactions at the molecular level. IEM-1460 binds to Ca2+-permeable AMPA-type glutamate
receptor channels and not to Ca2+-impermeable channels (Magazanik et al., 1997;
Tikhonov et al., 2000). This means that currents through AMPA-type channels are blocked by IEM-1460 except the channels contain a Q/R edited GluR2 subunit which renders the respective channels Ca2+-impermeable.
IEM-1460 was nearly ineffective at GluR2 flip GR channels which have a very low Ca2+-permeability. This specific effect of IEM-1460 was clearly confirmed by the results of our study and it holds also true when low amounts of cDNA of GluR2 flip GR subunits are used for coexpression at HEK293 cells. The IC50 of native human AMPA-type channels (except GluR2 flip GR channels) was in the range of low sensitivity rat hippocampal neurons (Magazanik et al., 1997).
In an additional study “Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels”, Krampfl et al. 2006, the two novel neuroprotective AMPA antagonists RPR119990 and RPR117824 were tested for their receptor interactions at the molecular level.
We could show that RPR119990 and RPR117824 block AMPA receptors competitively with an outstanding block activity as indicated by an IC50 in the low nanomolar range. The results hold true for different recombinant GluR channel subtypes. Especially the use of a non-desensitizing recombinant GluR channel enabled us to determine block mechanism and kinetics by co-application protocols.
An IC50 value in the low nanomolar range for AMPA receptor channels in combination with a reduced potency on kainate receptors makes these compounds extremely interesting for a therapeutic use, as relevant blood concentration might be achieved and future studies should settle the issue of AMPA-antagonism in clinical neuropharmacology.
Our studies point out the exceptional position of the GluR2 flip subunits in dependence of the resensitisation-kinetic and discuss possible consequences focused on the model of chronic excitotoxicity. Additionally the importance of electrophysiological studies of antagonist of AMPA-receptors as a supportive tool for drug screening was discussed.
6 Zusammenfassung
In den vorliegenden Arbeiten wurden elektrophysiologische Untersuchungen an rekombinanten AMPA-Typ Glutamatrezeptoren durchgeführt. AMPA-Rezeptoren sind für die exzitatorische synaptische Transmission im zentralen Nervensystem verantwortlich und stellen eine Untergruppe der ionotropen Glutamatrezeptoren dar.
Sie sind postsynaptisch lokalisiert, wobei Rezeptor und Ionenkanal eine funktionelle Einheit bilden. Durch die Bindung von Glutamat an AMPA-Rezeptoren wird eine für Kationen selektiv permeable Zentralpore geöffnet. Hierbei kommt es in Bruchteilen von Sekunden zu hohen Glutamatkonzentrationen im synaptischen Spalt.
Vier verschiedene Untereinheiten (GluR1-4) von AMPA-Rezeptoren sind bekannt.
Durch posttranskriptionelle mRNA-Editierung (R/G- und Q/R-Editierung) und alternatives Splicen (Flip- und Flop-Varianten) erhöht sich die Anzahl der verschiedenen Subtypen.
Die Q/R-Editierung existiert nur bei den GluR2-Untereinheiten und ist verantwortlich für die Regulation des intrazellulären Kalziumeinstroms. GluR2-Untereinheiten, die eine Editierung aufweisen sind impermeabel für Ca2+ -Ionen, während alle anderen Untereinheiten durchlässig für Kalziumionen sind. Die R/G-Editierung spielt eine wichtige Rolle in der Regulation der Desensitisierung und Resensitisierung. So zeigen editierte GluR2-Untereinheiten eine schnellere Kinetik in Bezug auf die Desensitisierung und Resensitisierung als die nicht-editierte GluR2-Untereinheit (Krampfl et al. 2002). Die Flip/Flop-Sequenz, die bei allen Untereinheiten vorkommt, ist ebenfalls an der Regulierung der Desensitisierung und Resensitisierung beteiligt, wobei die Flop-Varianten eine schnellere Desensitisierung als die Flip-Varianten aufweisen (Koike et al. 2000). AMPA-Rezeptoren kommen sowohl als homomere als auch als heteromere Rezeptoren vor.
Die in der vorliegenden Ph.D-These dargestellten Studie „Desensitization and resensitization are independently regulated in human recombinant GluR subunit coassemblies” , Schlesinger et al. 2005, wurden verschiedene AMPA- Rezeptor-Untereinheiten mit einander kombiniert und die Kinetik der einzelnen heterooligomeren Assemblierungen mit der Patch-Clamp-Technik untersucht. Hierbei konnte festgestellt werden, dass insbesondere die flip-Variante des GluR2 die Resensensitisierungskinetik dominiert, andere Parameter bleiben jedoch
unbeeinflusst. Orientierend an der Arbeit von Mansour et al. 2001 wurde aufgrund dieser Eigenschaften eine bevorzugte Assemblierung von Rezeptoruntereinheiten postuliert.
Darüber hinaus wurde in der Arbeit “Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors”, Schlesinger et al. 2005, die Wirkung des Adamantinderivates IEM-1460 an rekombinanten humanen homomeren AMPA-Rezeptoren sowie dem heteromeren GluR1o/2iGR-Rezeptor-Kanal untersucht. Die Experimente mit dem selektiven AMPA-Rezeptor-Antagonist IEM-1460 zeigten, dass die kalziumpermeablen homomeren GluR1o und GluR2iGQ eine signifikante Blockierung erfuhren. An dem kalziumimpermeablen homomeren GluR2iGR konnte kein signifikanter Effekt des IEM-1460 ausgemacht werden. Messungen am heteromeren GluR1o2iGR-Kanal zeigten ebenfalls keinen signifikanten Blockeffekt durch IEM-1460.
In einer weiteren Arbeit “Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels”, Krampfl et al. 2006, wurden AMPA-Typ Glutamatrezeptorantagonisten RPR119990 und RPR117824 sowie ihre Wechselwirkungen an rekombinanten Glutamatrezeptoren auf molekularer Ebene untersucht. Für beide Substanzen, RPR119990 und RPR117824, konnten in verschiedenen experimentellen Untersuchungen mögliche neuroprotektive Eigenschaften nachgewiesen werden (Canton et al.,2001; Mignani et al.,2002).
Bisherige pharmakologische Untersuchungen zeigten eine Wirksamkeit beider Substanzen als potente AMPA-Antagonisten im nanomolaren Konzentrationsbereich (Canton et al.,2001; Mignani et al.,2002).
Die Anwendung der Testsubstanzen am nicht-desensitiserenden AMPA-Rezeptor GluR2L504Y ermöglichte die weitere reaktionskinetische Charakterisierung der Blockmechanismen.
In Bezug auf die Pathophysiologie neurodegenerativer Erkrankungen, wie der amyotrophen Lateralsklerose, werden Desensitisierung, Resensitisierung und
Kalziumpermeabilität der AMPA-Rezeptoren als wichtige Faktoren diskutiert. Ins- besondere der Glutamat-vermittelten Exzitotoxizität, die zu einem unkontrollierten Ca2+ -Ionen-Einstrom führt und somit einen „Circulus vitiosus“ zur Folge hat, wird eine besondere Bedeutung beigemessen. Die Arbeiten heben zum einen die spezifische Eigenschaften heterooligomerer Assemblierungen von AMPA-typ Glutamatrezeptoren hervor und diskutieren ihre Bedeutung in Bezug auf die chronische Exzitotoxizität, zum anderen zeigen sie pharmakologische Angriffspunkte einer möglichen neuroprotektiven Therapie. Die Messdaten mit dem Adamantinderivat IEM-1460 zeigten eine selektive Blockierung kalziumpermeabler AMPA-Rezeptoren, was möglicherweise eine Therapiestrategie in der Behandlung neurodegenerativer Erkrankungen darstellen kann.
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Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors
*,1Friedrich Schlesinger, 1Derk Tammena, 1Klaus Krampfl & 1Johannes Bufler
1Neurological Department of the Medical School of Hannover, Karl-Neuberg Str. 1, 30623 Hannover, Germany
1 Antagonizing glutamatergic neurotransmission by blockade of AMPA-type glutamate receptors (GluR) is a promising pharmacological strategy for neuroprotection in neurodegenerative diseases and acute treatment of stroke.
2 We investigated the interaction of the adamantane derivative IEM-1460 with human wild-type and mutant AMPA-type GluR channels. Different recombinant homooligomeric human AMPA-type GluR channels and a rat nondesensitizing mutant GluR (GluR2L504Y) channel were expressed in HEK293 cells and investigated using the patch-clamp technique in combination with ultrafast agonist application.
3 When IEM-1460 was coapplied with glutamate, an open channel block mechanism was observed at slow desensitizing GluR2flip (X0.1 mMIEM-1460) and nondesensitizing GluR2L504Y channels (X1mMIEM-1460).
4 A competitive block of AMPA-type channels was observed with IC50 values for the dose block curves of 0.1 mMIEM-1460 at human unmutated and 10mMIEM-1460 at mutant GluR channels.
5 Nondesensitizing GluR2L504Y channels were used to further characterize the block mechanism.
After equilibration with the agonist, a current decay upon coapplication of glutamate and IEM-1460 was observed. The recovery from block was independent of the glutamate and IEM-1460 concentration. The extent of current inhibition as well as the time constant of current decay upon addition of the blocker to the test solution were dependent on agonist concentration; this strongly points to an additional competitive-like block mechanism of IEM-1460 at human AMPA-type GluR channels.
6 The data were interpreted in the frame of a molecular scheme with two binding sites of IEM-1460 at the receptor, one at the unliganded resting and the other at the fully liganded open state of the channels.
British Journal of Pharmacology(2005)145,656–663. doi:10.1038/sj.bjp.0706233 Published online 18 April 2005
Keywords: Patch clamp; ultrafast application; human AMPA-type receptors; block mechanism
Abbreviations: AMPA,a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; ALS, amyotrophic lateral sclerosis; DMEM, Dulbecco’s modified Eagle’s medium; GluR, glutamate receptor; HEK, human embryonic kidney; FCS, fetal calf serum; GFP, green fluorescent protein
Introduction
Glutamate is the major excitatory neurotransmitter of the central nervous system, which is toxic under certain patholo- gical conditions. Its main targets are ionotropic a-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), NMDA and kainate-type glutamate receptor (GluR) channels. Excessive release of glutamate is thought to participate in a pathophy- siological cascade, which leads to neuronal death in different acute neurological disorders like ischemic stroke and traumatic brain damage or in chronic neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) or Parkinson’s disease (Shaw, 1994; Williams et al., 1997; Dingledine et al., 1999).
Excitatory synaptic transmission is mediated mainlyviathe fast gating AMPA-type GluR channels and these channels are thought to play a crucial role in chronic neurodegeneration.
For example, glutamate-induced motor neuron death can be
prevented by blockade of AMPA-type GluR channels but not NMDA receptor antagonists (Vandenberghe et al., 2000;
Urushitaniet al., 2001). For that reason, targeting of human AMPA-type GluR channels is an interesting therapeutical principle in chronic neurodegenerative disorders but despite proven neuroprotective properties in cell culture assays, none of the AMPA-type GluR channel antagonists has so far passed clinical testing (Dingledineet al., 1999).
Adamantane derivatives such as IEM-1460 block AMPA- and NMDA-type GluR channels. IEM-1460 was especially used to identify Ca2þ-permeable AMPA-type receptors in the central nervous system, because it interacts specifically with these channels. The action of IEM-1460 at AMPA-type GluR channels was interpreted as an open channel block (Magazanik et al., 1997). In the present study, we investigated the effect of IEM-1460 on different recombinantly expressed wild-type and nondesensitizing mutant GluR channels and could show that this drug blocks exclusively Ca2þ-permeable GluR channels, that is, GluR channels that do not contain GluR2R subunits,
*Author for correspondence:
British Journal of Pharmacology(2005) 145,656–663 &2005 Nature Publishing Group All rights reserved 0007 – 1188/05$30.00 www.nature.com/bjp
