Elsevier

Neuropharmacology

Volume 37, Issues 10–11, 1 October 1998, Pages 1239-1247
Neuropharmacology

Antagonism of neuronal kainate receptors by lanthanum and gadolinium

https://doi.org/10.1016/S0028-3908(98)00082-3Get rights and content

Abstract

The effects of lanthanum and gadolinium on currents evoked by excitatory amino acids were studied in cultured rat hippocampal and cortical neurons, in freshly dissociated dorsal root ganglion neurons, and in human embryonic kidney 293 cells expressing the GluR6 kainate receptor subunit. In all of these cells, currents mediated by kainate-preferring receptors were antagonized by low micromolar concentrations of the trivalent ions. At negative holding potentials, the IC50 values for inhibition in DRG cells were 2.8 μM for La and 2.3 μM for Gd. Kainate receptor-mediated currents in hippocampal neurons and in 293 cells expressing GluR6 were blocked by La with IC50 values of 2.1 and 4.4 μM, respectively. Steady-state inhibition by the lanthanides showed very slight dependence on membrane potential, however, we were not able to resolve any systematic variation with membrane potential in the kinetics of block onset or recovery. Inhibition was not use-dependent and was not overcome by increasing the concentration of agonist. These results indicate that lanthanides probably do not bind deep within the ion pore or directly compete for the agonist binding site. In contrast to neuronal AMPA receptors, which require more than 100 μM lanthanides for half-maximal blockade, the inhibition of neuronal and recombinant kainate receptors by these ions displays significantly higher potency.

Introduction

The amino acid glutamate activates three different families of ion channels that are named for the agonists N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate (Collingridge and Lester, 1989). Previous work has demonstrated that NMDA and AMPA receptors underlie the rapid depolarization of postsynaptic neurons at fast excitatory synapses throughout the central nervous system (Monaghan et al., 1989). More recently, kainate receptors have attracted wide attention as potential mediators of both presynaptic (Chittajallu et al., 1996, Clarke et al., 1997, Rodriguez-Moreno et al., 1997) and postsynaptic (Castillo et al., 1997, Vignes and Collingridge, 1997) responses in the rat hippocampus. In addition, all three receptor subtypes have been implicated in the neuronal damage associated with ischemia and other disorders (Monaghan et al., 1989).

Detailed analysis of neuronal kainate receptors was originally restricted to freshly dissociated dorsal root ganglion (DRG) neurons, which do not express functional NMDA or AMPA receptors (Huettner, 1990, Wong and Mayer, 1993). In particular, the fact that most agonists (Wong et al., 1994) and antagonists (Wilding and Huettner, 1996) of non-NMDA receptors display relatively little selectivity between AMPA and kainate-preferring receptors made it difficult to distinguish the relative contributions of these two subtypes in neurons that expressed both. More recently, the discovery of compounds such as GYKI 53655 (Donevan et al., 1994), which produce potent, selective, non-competitive block of AMPA receptors has made it possible to dissect out the currents that flow through kainate receptor channels in CNS neurons (Paternain et al., 1995, Wilding and Huettner, 1997). As expected from work on DRG cells (Huettner, 1990, Wong and Mayer, 1993) and recombinantly expressed kainate receptor subunits (Herb et al., 1992, Sommer et al., 1992), currents mediated by kainate receptors in hippocampal neurons (Lerma et al., 1993, Wilding and Huettner, 1997) undergo strong desensitization, recover from desensitization very slowly, and are activated by low micromolar concentrations of kainate.

In order to characterize better the pharmacological differences between native AMPA and kainate receptors, we have examined the action of divalent and trivalent cations on currents mediated by these receptors in cultured or freshly dissociated neurons. A number of previous studies have demonstrated that excitatory amino acid receptors exhibit differential sensitivity to inhibition by metal ions including magnesium (Mayer et al., 1984, Nowak et al., 1984), zinc (Peters et al., 1987, Westbrook and Mayer, 1987) and lanthanum (Reichling and MacDermott, 1991). Because much of this earlier work focused on NMDA and AMPA receptors, however, relatively little is known about the sensitivity of the kainate receptor subtype to inhibition by metal ions. In the present study, we have followed up on a preliminary observation that kainate receptors expressed by hippocampal (Wilding and Huettner, 1997) and primary sensory neurons (Huettner, 1991) are significantly more sensitive than AMPA receptors to blockade by lanthanum (La) and gadolinium (Gd).

Section snippets

Cell preparation

DRG neurons were dissociated by trituration following incubation with protease XXIII (Sigma, St. Louis, MO) as described by Wilding and Huettner (1995). Freshly dissociated cells were maintained overnight at room temperature in Earl's balanced salt solution (EBSS) until used the following day. Hippocampal and cortical neurons from 2- to 6-day-old Long–Evans rats were dissociated with papain (Huettner and Baughman, 1986, Wilding and Huettner, 1997). Hippocampi from two rat pups were cut into

Selective inhibition of kainate current in DRG cells

Whole-cell currents activated by 200 μM kainate were used to study the action of a series of metal cations on kainate and AMPA receptors. Kainate receptors were studied in freshly dissociated DRG neurons that had been exposed to concanavalin A in order to block desensitization (Huettner, 1990). Currents mediated by AMPA receptors were recorded in cultured rat cortical and hippocampal neurons. Numerous previous studies have demonstrated that kainate evokes large maintained currents through AMPA

Relative potency

This study has demonstrated the potent inhibition by trivalent lanthanides, La and Gd, of whole-cell currents mediated by kainate receptors. Inhibition was half-maximal at 2–4 μM in freshly dissociated DRG cells, in cultured hippocampal neurons and in HEK 293 cells transfected with the GluR6 subunit. A very similar IC50 (2 μM) was obtained by Reichling and MacDermott (1991)for blockade of NMDA receptor-mediated currents in dorsal horn neurons. In contrast, inhibition by lanthanides is somewhat

Acknowledgements

This work was supported by the NIH (Grant NS30888) and by the McDonnell Center for Cellular and Molecular Neurobiology. We are grateful to Dr Steven Heinemann for providing the clone of GluR6, to Dr David Clapham for providing the vector containing L3T4, to Dorothy Turetsky for HEK cells, to Eli Lilly and Company for LY300168 (GYKI 53655), and to our colleagues in the Department of Cell Biology and Physiology for advice on molecular biology.

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