Regulation of AMPA receptor dephosphorylation by glutamate receptor agonists
Introduction
The neostriatum, including the dorsal striatum and the nucleus accumbens, plays a critical role in regulating functions as diverse as the control of motor activity (Albin et al., 1989) and the development of drug addiction (Nestler, 2001). Ascending dopamine (DA)-containing neurons from the midbrain innervate the predominant cell type of the striatum and nucleus accumbens, the medium spiny-type neuron (Kemp and Powell, 1971). DA, released from these neurons, modulates the excitability of medium spiny neurons in response to glutamate, released from afferents from the cortex and other brain regions.
Three subtypes of ligand-gated ion channels, N-methyl-d-aspartate (NMDA), (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainate subclasses, mediate actions of glutamate in the brain (Hollamnn and Heinemann, 1994). Each of these receptor subtypes is expressed in medium spiny neurons of the striatum and nucleus accumbens (Petralia and Wenthold, 1992, Martin et al., 1993, Tallaksen-Greene and Albin, 1994, Ghasemzadeh et al., 1996, Bernard et al., 1997).
AMPA receptors, in particular, are believed to mediate a large proportion of glutamatergic excitation of medium spiny neurons (Cepeda et al., 1993). Phosphorylation of the GluR1 subunit of the AMPA receptor at Ser845 by cAMP-dependent protein kinase (PKA) (Roche et al., 1996) and at Ser831 by protein kinase C (PKC) or Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Barria et al., 1997) enhances the activity of AMPA channels. Moreover, the increased phosphorylation of GluR1 at Ser845 (Snyder et al., 2000) in response to D1-type DA receptor activation has been implicated in the enhancement of AMPA receptor currents in medium spiny neurons (Yan et al., 1999). The ability of DA to regulate both GluR1 phosphorylation and AMPA channel activity requires the inhibition of protein phosphatase 1 (PP1), a major protein serine/threonine phosphatase in the brain. Inhibition of PP1 activity is mediated via a biochemical cascade involving the phosphorylation by PKA of dopamine- and cyclic AMP (c-AMP)-regulated phosphoprotein, Mr 32,000 (DARPP-32), a protein that is highly enriched in medium-sized spiny neurons (Ouimet et al., 1984). DARPP-32, when phosphorylated by PKA on a single threonine residue (Thr34), is converted into a potent inhibitor of PP1 (Hemmings et al., 1984). As a result, DARPP-32 plays a critical role in regulating the excitability of medium spiny neostriatal neurons through its functional regulation of a number of cellular effectors. In addition to AMPA receptors, these include voltage-dependent Ca2+ and Na+ channels, the electrogenic ion pump Na+, K+-ATPase, and NMDA and GABAA receptors (Fienberg et al., 1998, Greengard et al., 1999, Snyder et al., 2000, Flores-Hernandez et al., 2000).
It has been shown that activation of NMDA receptors in a model of hippocampal long-term depression (LTD) results in a marked dephosphorylation of GluR1 at Ser845 (Lee et al., 1998) suggesting that glutamate-induced dephosphorylation of GluR1 may be a critical mechanism for regulating the functional activity of AMPA receptors. Activation of NMDA receptors on striatal neurons has been shown to increase Ca2+ influx into neurons (MacDermott et al., 1986), leading to the dephosphorylation of DARPP-32 by calcineurin, the Ca2+/calmodulin-dependent protein phosphatase (Halpain et al., 1990). The aim of the present study was to determine whether glutamate, via activation of NMDA and/or non-NMDA subclasses of glutamate receptors, might also regulate the dephosphorylation of GluR1 in neostriatal neurons and whether this might involve DARPP-32 and PP1.
Section snippets
Materials
(±)-α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA hydrobromide), 6-cyano-7-nitroquinoxaline-2,3-dione, disodium salt (CNQX), N-methyl-D-aspartate, d(−)-2-amino-5-phosphonopentanoic acid (AP-5), and MK-801 hydrogen maleate were obtained from Research Biochemicals, Inc. (Natick, MA) and Sigma Chemical Co. (St. Louis, MO). Nifedipine and cyclosporin A were purchased from Alexis Biochemicals (San Diego, CA). Goat anti-mouse IgG was purchased from Pierce, nitrocellulose
AMPA-induced dephosphorylation of GluR1 at Ser845: calcineurin-dependence
The regulation of phosphorylation of Ser845, a site that controls channel open time probability (Roche et al., 1996), was examined in nucleus accumbens slices. The GluR1 subunit of the AMPA receptor was detected as a band of Mr~106 kDa in homogenates of rat nucleus accumbens (Fig. 1A). A basal level of phosphorylation was detected in untreated slices. Treatment of slices with AMPA, an agonist at AMPA and kainate subclasses of glutamate receptors, promoted a rapid dephosphorylation of Ser845
Discussion
The present study demonstrates that the activation of AMPA/kainate subclass of glutamate receptor, an expected result of D1-mediated phosphorylation of GluR1, induces a rapid dephosphorylation of GluR1 at Ser845 and of DARPP-32 at Thr34. However, dephosphorylation of GluR1 in response to AMPA did not appear to require regulation of PP1 activity. This conclusion is based on the inability of calyculin A, at concentrations that are known to block most PP1 activity in slices, to prevent
Acknowledgements
These studies were supported by U.S.P.H.S. Grant 40899 (to P.G., G.L.S., and A.C.N.). The authors wish to thank Dr. Akinori Nishi for helpful comments and Carmina Valle and Peter Ingrassia for excellent technical assistance.
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