Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro

Abstract

The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 μM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC₅₀ values of 1.77 μM (GluR1_i), 0.22 μM (GluR2_i), 0.56 μM (GluR2_o), 1.89 μM (GluR3_i) and 0.20 μM (GluR4_i) for LY392098 and EC₅₀ values of 5.65 μM (GluR1_i), 0.15 μM (GluR2_i), 1.44 μM (GluR2_o), 1.66 μM (GluR3_i) and 0.21 μM (GluR4_i) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3–10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells 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).