Coexpression of N-methyl-D-aspartate and phencyclidine receptors in Xenopus oocytes injected with rat brain mRNA.

Abstract

Recent evidence suggests that the N-methyl-D-aspartate (N-Me-D-Asp) channel is functionally and structurally associated with the phencyclidine (PCP) receptor, which mediates the psychotomimetic effects of PCP, a opioids, and dioxalanes. To investigate the relationship between N-Me-D-Asp and PCP receptors on a molecular level, we injected mRNA isolated from adult rat brain into Xenopus oocytes. In injected oocytes N-Me-D-Asp application (with glycine) evoked a partially desensitizing inward current that was potentiated by glycine and blocked by D-(-)-amino-5-phosphonovaleric acid (D-APV), by Zn2' and, in a voltage-dependent manner, by Mg2 . These results show that the distinguishing features ofrat brain N-Me-D-Asp channels are reproduced in this translation system. In addition, kainic acid elicited a nondesensitizing inward current at short latency, and quisqualate elicited a delayed oscillatory inward current, presumably mediated by a second-messenger system. Responses to glutamate had both short-latency and delayed components. (21) Autoradiographic studies demonstrate that the two receptors are colocalized in rat forebrain and midbrain (24,25). However, N-Me-D-Asp receptors occur independently of PCP receptors in the cerebellum (25).The Xenopus oocyte is a self-contained expression system that is particularly suitable for the study of the relationship between receptor structure and function and for identifying the mRNA that directs receptor synthesis. This system has been shown to faithfully translate neuroreceptor (23,(26)(27)(28) and channel (29,30)

Purified unitary kainate/alpha-amino-3-hydroxy-5-methylisooxazole-propionate (AMPA) and kainate/AMPA/N-methyl-D-aspartate receptors with interchangeable subunits.

“…We suggest that this situation extends further, that subunit interchanges can also occur between the non-NMDA and certain NMDA subunits, and that the resultant oligomers again form a common channel. A model that explains our results and others reviewed above (3,22,23,26 The subunit size difference in Xenopus renders this situation more readily detected; however, we have seen similar effects in rat brain poly(A) RNA oocyte expression. Such an exchange, if it produced a stable oligomer with two 42-kDa and three 100-kDa subunits, would account for the size difference seen (Fig.…”

“…The NMDA-evoked currents and their blockade by MK-801 and Mg2+ were as reported for oocytes injected with rat brain RNA (22,23). When KA (0.1 mM) was applied as a pulse (10 s) during perfusion of an oocyte with AMPA (0.1 mM), the response to the KA was reduced by r90%.…”

“…It could be envisaged that NMDA is a competitive desensitizing agonist [as is AMPA (22,23)] or is a weak antagonist at the KA receptor. This was excluded because the observed effect is distinctly noncompetitive (Fig.…”

Ca2+Influx Amplifies Protein Kinase C Potentiation of Recombinant NMDA Receptors

“…Adult female Xenopus laevis (Xenopus I, Ann Arbor, M I) were anesthetized by immersion in ice water or 0.15% aminobenzoic acid ethyl ester, and oocytes were isolated and prepared as described (Kushner et al, 1988(Kushner et al, , 1989. Selected stage V and V I oocytes were injected with in vitro transcribed RNA (ϳ20 ng /cell; for heteromeric receptor expression, N R1 and N R2 were mixed in a ratio of 1:3) and maintained at 18°C in culture buffer (in mM: 103 NaC l, 2.5 KC l, 2 MgC l 2 , 2 CaCl 2 , and 5 H EPES, pH 7.5).…”

Characterization of Two Novel N-Methyl-D-aspartate Antagonists: EAA-090 (2-[8,9-Dioxo-2,6-diazabicyclo [5.2.0]non-1(7)-en2-yl]ethylphosphonic Acid) and EAB-318 (R-α-Amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic Acid Hydrochloride)

“…Oocytes were collected from anesthetized Xenopus laevis as described previously (Kushner et al, 1988). After removing the follicular layer, stage V and VI oocytes were injected with in vitrotranscribed RNA (about 20 ng/cell) and maintained at 18°C in culture buffer (103 mM NaCl, 2.5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 5 mM HEPES, pH 7.5).…”