Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro
Introduction
Glutamate is the primary excitatory neurotransmitter in the central nervous system and can act at three classes of ionotropic glutamate receptors (Collingridge and Lester, 1989, Bleakman and Lodge, 1998) named on the basis of agonist pharmacology as N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA). The molecular cloning of rat AMPA receptor subunits, GluR1-4, has allowed studies to be performed on the pharmacological and biophysical properties of these ion channels in recombinant expression systems (Hollmann and Heinemann, 1994). The human AMPA receptor ion channel subunits, GluR1, GluR2, GluR3 and GluR4, have also been cloned and expressed in HEK293 cells (Hoo et al., 1994, Korczak et al., 1995, Fletcher et al., 1995, Bleakman et al., 1999, Clarke et al., 1997, Bortolotto et al., 1999). For rat AMPA receptors it is thought that 4 or 5 subunits assemble to form a functional ion channel complex. The pharmacological and biophysical properties of AMPA receptors ion channels appear to be determined by the presence or absence of subunits in the ion channel complex. For example, the presence of a GluR2 subunit into the ion channel hetero-oligomer results in reduced calcium permeablility (Hollmann et al., 1991) an effect due to RNA editing of a Q/R site in the GluR2 RNA (Sommer et al., 1991). Additional diversity of AMPA receptor subunits is conferred on the ion channels by the existence of two isoforms, flip and flop, for each subunit (Sommer et al., 1990). The two isoforms differ with regard to amino acid structure in a 38 amino acid sequence in the TMIII-IV region and differ functionally with regard to desensitization profiles with “flip” (i) isoforms desensitizing less rapidly than “flop” (o) isoforms. Rates of recovery from desensitization are reported to be affected by editing of an R/G site within RNA's encoding GluR2, GluR3 and GluR4 (Lomeli et al., 1994).
Compounds that have been described to enhance ion influx through AMPA receptors include the nootropic pyrrolidones such as aniracetam, oxiracetam, piracetam and related compounds such as CX516 (Ito et al., 1990, Staubli et al., 1990, Copani et al., 1992, Arai et al., 1994). The benzothiadiazides such as cyclothiazide, diazoxide, IDRA-21, S18986-1 (Vyklicky et al., 1991, Yamada and Rothman, 1992, Thompson et al., 1995, Desos et al., 1996) and the compound 4-[2-(phenylsulfonylamino)thio]-difluor-phenoxyacetamide (PEPA) (Sekiguchi et al., 1997) are also compounds that have been shown to positively modulate AMPA receptors. Several studies support the potential utility of positive allosteric modulators of AMPA receptors in the treatment of cognitive deficits. For example, IDRA-21 has been demonstrated to improve learning in rats (Zivkovic et al., 1995) and monkeys (Thompson et al., 1995). In addition CX516 has been shown to improve memory retention and performance in aged rats (Granger et al., 1996) and memory encoding in humans (Ingvar et al., 1997).
Positive allosteric modulators of AMPA receptors appear to enhance AMPA receptor-mediated responses by reducing the extent to which receptor desensitization occurs (Partin et al., 1996, Partin et al., 1995; Sekiguchi et al., 1997). The ability of AMPA receptor modulators to enhance AMPA currents is in part determined by splice variant composition of AMPA receptors. For example, it has been demonstrated that aniracetam and cyclothiazide are more effective in potentiating flop and flip isoforms, respectively (Partin et al., 1994, Johansen et al., 1995), whereas PEPA is more effective at the flop isoform (Sekiguchi et al., 1997).
The current study has investigated the ability of two novel compounds, LY392098 and LY404187 (Fig. 1), to potentiate AMPA receptor-mediated responses of recombinant human AMPA receptor subunits expressed in HEK293 cells. In particular, we have examined the potency and selectivity of the two compounds to potentiate the responses of the four recombinant human AMPA receptor subtypes. Comparisons have also been made with two other AMPA receptor potentiators, cyclothiazide and CX516. In addition, the effects of cyclothiazide, LY392098 and LY404187 on AMPA receptor responses have also been examined at human GluR4i receptors using whole cell electrophysiological recordings. A preliminary report of this work has been presented (Bleakman et al., 2000).
Section snippets
Transfected cell lines
Stable cell lines of HEK293 cells transfected with cDNA coding for the human GluR1-4 were established as reported previously (Fletcher et al., 1995, Clarke et al., 1997). Stable cell lines incorporating the cDNA for human GluRs were established by transfection of HEK293 cells with the mammalian expression vector pRc/CMV (Invitrogen). Transfected cells were selected on the basis of G418 resistance, and expression of both genes confirmed by RT-PCR.
Calcium influx measurements
96-well plates containing confluent monolayers of
Calcium influx studies
In the absence of an AMPA receptor potentiator, glutamate was without effect upon basal calcium levels (as determined by changes in relative fluorescence). However, cyclothiazide, LY392098 and LY404187 (Fig. 1) produced a rise in cellular calcium levels in the presence of glutamate (100 μM). We also examined the effect of NBQX (50 μM) on the increase in fluorescence observed for glutamate (100 μM) for GluR1-4 flip and GluR2 flop expressing cells in the presence of cyclothiazide, or LY404187. In
Discussion
Allosteric modulation of AMPA receptor-mediated responses has been described in a number of previous studies. For example negative allosteric modulators of AMPA receptors include the 2,3 benzodiazepine series of compounds (Bleakman et al., 1996) and positive allosteric modulators include compounds such as the pyrrolidones, benzothiadiazides and more recently the compound 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA) (Ito et al., 1990, Isaacson and Nicoll, 1991,
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2012, Neuroscience LettersCitation Excerpt :The second possibility is the difference in the series resistance between the two studies: the series resistance in the study by Choi et al. [5] was 7.3 ± 0.3 MΩ, and the series resistance in the study by Montgomery et al. was 14.1 ± 2.2 MΩ. The series resistance could set the detection limit for synaptic responses, especially for synapses far from the cell soma, and hence, the relatively high series resistance in the study of Montgomery et al. [13] may have hampered the detection of small but slow AMPA EPSCs that may have been revealed in the presence of cyclothiazide. In summary, this study has characterized the effects of LY404187 on pre- and postsynaptic functions at CA3–CA1 synapses in neonatal rats.
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