Elsevier

Neuropharmacology

Volume 41, Issue 6, November 2001, Pages 650-663
Neuropharmacology

Mechanism and impact of allosteric AMPA receptor modulation by the AmpakineTM CX546

https://doi.org/10.1016/S0028-3908(01)00133-2Get rights and content

Abstract

Glutamate release at central synapses is transduced into a characteristic fast postsynaptic response by AMPA receptor gating and agonist affinity. The effect of two classes of modulators of AMPA receptor desensitization, the benzothiadiazides (cyclothiazide and IDRA 21) and the benzoylpiperidines (CX516 and CX546), were studied on gating kinetics of recombinant, native AMPA receptors and on synaptic currents. CX546 reduced the degree of desensitization more potently than CX516 or IDRA 21, but not as efficiently as cyclothiazide. In presence of CX516/CX546, desensitization of GluR2flip receptors was inhibited more than of GluR1flip, whereas they had no effect upon response shape or conductance. CX546 increased agonist affinity threefold on nondesensitizing AMPA receptors by slowing agonist unbinding. Analysis of modulatory action suggests that, in contrast to cyclothiazide or IDRA 21, the AmpakineTM CX546 binds specifically to the agonist bound nondesensitized receptor, most likely acting by destabilizing the desensitized receptor conformation. All modulators tested showed higher efficiency on native receptors as compared to homomeric receptors. At the glutamatergic synapse, evoked synaptic amplitudes were weakly potentiated, while EPSC decay was slowed by nearly a factor of three in the presence of CX546 or cyclothiazide. In the presence of CX546, the current induced by short pulses of glutamate from recombinant GluR2 receptors decayed with a time course that was approximately twentyfold faster than EPSCs. The unique properties of CX546 may be beneficial for therapeutical use.

Introduction

Fast excitatory synaptic transmission in the central nervous system is mediated predominantly by Ī±-amino-3-hydroxy-5-methyl-4-isooxazolepropionic acid (AMPA) type glutamate receptors. The importance of this receptor family in brain function and potential downregulation of their functions in numerous diseases has triggered a strong interest to find drugs that interfere with its gating properties during synaptic transmission. Ubiquitous expression of AMPA receptors however have made it sofar difficult to identify modulators that act on specific function of the nervous system such as memory, cognition and other neurological states. Heteromeric assembly of the AMPA receptor subunits GluR1-4 form ionotropic ion channels that can transduce the presynaptic signal into a surprisingly variable postsynaptic response. The subunit composition varies with particular brain region, with the cell type and during development. Further posttranslational modification of the AMPA receptor gene alters channel gating and permeability in a distinct fashion, mainly influencing receptor deactivation and desensitization. Whilst their individual contributions are debated, these two parameters are thought to be the crucial parameters that shape the synaptic response.

Recent structural and functional data provide detailed insights into both the intricate molecular mechanisms underlying activation, deactivation and desensitization of AMPA receptors and how the gating properties are modulated by drugs (Armstrong and Gouaux, 2000, Partin, 2001, Stern-Bach et al., 1998). A number of allosteric modulators that affect the process of desensitization and deactivation have been characterized in detail. These include the benzothiadiazides such as cyclothiazide, diazoxide and IDRA 21 (Bertolino et al., 1993, Patneau et al., 1993, Yamada and Rothman, 1992, Yamada and Tang, 1993), the pyrrolidinone analogs aniracetam, piracetam (Gouliaev and Senning, 1994, Isaacson et al., 1991, Partin et al., 1996, Tang et al., 1991), and a heterologous group of substances named ampakines, whose properties have been characterized to modify AMPA receptor function. Ampakines include modulators such as BCP, CX516, CX546 (Arai et al., 1996c, Holst et al., 1998, Lauterborn et al., 2000, Lynch, 1998). Most modulators are relatively specific for AMPA-type glutamate receptors which also, in part, affect splice variant (flip/flop) differentially (Partin et al., 1995, Partin et al., 1996).

Among benzothiadiazides, cyclothiazide is one of the most potent modulators that is best understood in terms of its mechanism of action (Partin, 2001, Partin et al., 1996, Partin et al., 1994, Partin et al., 1993, Yamada and Tang, 1993). IDRA 21, being less potent than cyclothiazide but more potent than diazoxide and aniracetam (Bertolino et al., 1993), has gained strong interest because of its non-neurotoxic effects in primary cultures, potency in abating pharmacologically induced cognitive impairments in patas monkeys (Impagnatiello et al., 1997, Thompson et al., 1995), cognition enhancement in rats (Zivkovic et al., 1995) and promoting the induction of long term potentiation (LTP) (Arai et al., 1996a). On the negative side, recent reports (Yamada et al., 1998) show that IDRA 21 worsens neuronal ischemic injury due to excessive AMPA receptor activation.

Ampakines are molecular entities that have shown their importance as positive modulators in enhancing memory of rodents and humans (Granger et al., 1996, Ingvar et al., 1997, Larson et al., 1995). The potential significance of ampakines in their therapeutic effectiveness arises due to their ability to freely cross the bloodā€“brain barrier (Staubli et al., 1994b), promote LTP induction in vivo (Lynch, 1998), improve short term memory in radial maze (Staubli et al., 1994a), facilitate olfactory learning (Larson et al., 1995), and effectively boost transmission in complex neuronal networks (Sirvio et al., 1996). Ampakines like BDP20 (Arai et al., 1996b), CX614 (Arai et al., 2000) and BDP12 (Arai et al., 1996c) are apparently without direct agonistic or antagonistic properties and do not affect the membrane potential or inhibitory transmission to a detectable extent (Arai et al., 1996c). They are thought to act by modulating the kinetics of desensitization and deactivation (channel closing and transmitter dissociation), resulting in a modified shape of excitatory synaptic responses (Arai et al., 1996b, Arai and Lynch, 1998). Binding tests on AMPA subunits have shown that some ampakines like CX614 or BDP-37 apparently do not have major preferences for flip/flop splice variants (Arai et al., 2000, Hennegriff et al., 1997).

In the current investigation, we have examined the pharmacological action of structurally distinct modulators IDRA 21 (a benzothiadiazine derivative) and the ampakines CX516 and CX546 (benzoylpiperidine derivatives) on flip version of wild-type GluR1/2, the nondesensitizing GluR1L497Y, native receptors and on synaptic transmission. Most analysis is concerned with the action of CX546, a more potent analog than CX516. We compared the less characterized basic kinetic properties of their modulatory action to gain insight into the molecular mechanism during channel gating and desensitization process. Our results on recombinant and native receptors show a striking difference in the degree of modulation based on the subunit composition as well as in their apparent mode of action. All drugs tested modulated excitatory synaptic currents by moderately enhancing the peak amplitude and slowing down EPSCs, although with differing efficacy.

Section snippets

Plasmids and mutagenesis

cDNAs encoding the alternatively spliced flip version of wild type GluR1Q and GluR2Q (Sommer et al., 1990) were used for all the experiments. Point mutations were introduced as described (Stern-Bach et al., 1998). All receptors were subcloned into pRK5 vectors (InVitrogen). Amino acid numbering starts from the first methionine of the open reading frame.

Cell culture and transfections

Cells from the human embryonic kidney cell line HEK293 (ATCC, USA) were cultured in RPMI 1640 (Gibco BRL) supplemented with 10% fetal calf serum

Results

We first describe the basic modulatory action of the benzoylpiperidines CX546 and CX516 and the benzothiadiazides IDRA 21 (Fig. 1) on a defined AMPA receptor population. We used flip splice variants of wild-type GluR1 and also the unedited large conducting GluR2Q receptor to probe for the potential differences between subunits. The subunits were transiently transfected in HEK293 cells (Chen and Okayama, 1988) and outside-out patches were exposed to control and agonist solution in the absence or

Discussion

The goal of this study was to examine the mechanism of two exemplary AMPA receptor modulators of a functionally defined class, the ampakines CX546 and CX516 (benzoylpiperidine derivatives), and compare these with a well studied class of benzothiadiazide modulators such as cyclothiazide and IDRA 21. On recombinant or native AMPA receptors, CX546 reduced the degree of desensitization more potently than CX516 or IDRA 21, but not as efficiently as cyclothiazide. We noted larger absolute

Acknowledgments

The authors thank Cortex Pharmaceuticals Inc. for providing the ampakines CX516 and CX546 used in this study, Dr. Peter H. Seeburg and Dr. Yael Stern-Bach for cDNA clones and Ina Herfort for technical assistance.

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