Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 9971-9974, December 1990 Neurobiology Polyamines potentiate responses of N-methyl-D-aspartate receptors expressed in Xenopus oocytes (glutamate/excitatory neurotransmitter/spermine/synaptic transmission) JAMES F. MCGURK, MICHAEL V. L. BENNETT, AND R. SUZANNE ZUKIN Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 Contributed by Michael V. L. Bennett, September 19, 1990 ABSTRACT Glutamate, the major excitatory neurotrans- tiate the NMDA response. Moreover, spermine increased the mitter in the central nervous system, activates at least three affinity of the receptor for glycine without affecting its inter- types of channel-forming receptors defined by the selective action with NMDA (31, 32). agonists N-methyl-D-aspartate (NMDA), kainate, and quis- Polyamines have been shown to have a range of effects on qualate [or more selectively by a-amino-3-hydroxy-5-methyl- responses of glutamate receptors in hippocampal neurons 4-isoxazolepropionic acid (AMPA)]. Activation of the NMDA (33) and in Xenopus oocytes injected with messenger RNA receptor requires glycine as well as NMDA or glutamate. (mRNA) from rat and chicken brain (34). Spermine potenti- Recent studies have provided evidence that certain polyamines ated NMDA-induced currents in hippocampal neurons while potentiate the binding by NMDA receptors of glycine and the 1,10-diaminodecane decreased them; diethylenetriamine had open channel blocker MK-801. To determine whether poly- no action on NMDA responses but antagonized actions of amines alter channel opening, we examined their effects on rat both spermine and 1,10-diaminodecane, which may therefore brain glutamate receptors expressed in Xenopus oocytes. Our be considered positive and negative allosteric modulators results demonstrate that spermine potentiates the response of (33). In Xenopus oocytes, currents induced by NMDA, the NMDA receptor but has no effect on responses to kainate kainate, and quisqualate were potentiated by spermine (34). and quisqualate. Furthermore, spermine increases the maxi- Polyamines are normal constituents of cells and play mum response to NMDA and glycine and acts, at least in part, essential roles in cell proliferation and differentiation (35). by increasing the apparent affinity of the NMDA recep- Although they may have some influence on neurotransmis- tor/channel complex for glycine. The present rindings and the sion (36) and excitability of neurons during epileptiform fact that polyamines are a natural constituent of brain suggest kindling (37) and their synthesis may be regulated by neu- that polyamines may play a role in the regulation of gluta- ronal activity (38), their role, if any, in modulation of NMDA matergic transmission. receptors in the central nervous system is unknown. The experiments described here were undertaken to ex- amine the pharmacology of modulation of glutamate recep- The N-methyl-D-aspartate (NMDA) receptor is a ligand- tors by selected polyamines. We utilized receptors expressed gated channel that mediates a slow response to glutamate in in Xenopus oocytes injected with rat brain mRNA. This neurons of the central nervous system (1-3). An understand- expression system is well suited to the study of receptor- ing of the pharmacology of agents that modulate activity of ligand interactions, since it can translate channel-forming the NMDA receptor is important because ofthe putative role proteins from exogenous mRNA and provide necessary of the receptor in central nervous system processes such as posttranslational modifications including phosphorylation, long-term potentiation (4-6), learning (7), developmental glycosylation, and assembly into the membrane. Exogenous structuring (8-11), excitotoxicity, ischemic cell death (12- excitatory amino acid receptors expressed in oocytes have 15), epilepsy, kindling (16, 17), Huntington disease (12, 18), been shown to have the pharmacological and biophysical and schizophrenia (19). characteristics of receptor proteins in neurons (39, 40). Evidence from pharmacological and physiological studies indicates that the NMDA receptor has a number ofregulatory domains. Glutamate and glycine have distinct binding sites on MATERIALS AND METHODS the NMDA receptor, both of which must be occupied for the RNA was extracted from brains rostral to the brainstem of channel to open (20-23). Both phencyclidine receptor ligands adult male Sprague-Dawley rats by a guanidinium isothio- and Mg2' block NMDA-induced currents in a voltage- cyanate/cesium chloride density gradient centrifugation dependent manner, suggesting that they bind within the method as described (22, 41, 43, 44). Poly(A)+ RNA was then lumen ofthe channel (24, 25). Zinc inhibits NMDA responses purified by oligo(dT)-cellulose chromatography, dissolved in (26, 27), primarily by binding at a distinct site outside of the water, and stored at -70'C until use. Oocytes were removed channel. It also appears to be a low-affinity channel blocker, from the ovarian lobes of anesthetized Xenopus laevis. much less effective than Mg2+ (28). Following incubation for 2 hr in Ca2l-free ND-96 medium Recent evidence from receptor binding studies indicates (82.5 mM NaCl/2 mM KCl/1 mM MgCl2/5 mM Hepes that there is still another regulatory site on the NMDA buffer, pH 7.5) containing collagenase (2 mg/ml), penicillin receptor/channel complex. The polyamines N,N'-bis(3- (100 units/ml), and streptomycin (1 mg/ml), the follicular aminopropyl)-1,4-butanediamine (spermine) and N-(3-ami- layer was removed manually. Stage V and VI oocytes (45) nopropyl)-1,4-butanediamine (spermidine) increased binding were then selected and injected with RNA (50 ng per oocyte). of the potent phencyclidine receptor ligand [3HJMK-801 (29, Oocytes were maintained for 5-9 days at 18'C in Leibovitz's 30) and of [3H]glycine (31). These results and similar findings L-15 medium supplemented with penicillin (100 units/ml) and by others (32) suggested that certain polyamines might poten- streptomycin (1 mg/ml) and buffered with Hepes (5 mM, pH 7.6). Electrophysiological recordings took place in a chamber The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: NMDA, N-methyl-D-aspartate; Imax, maximum cur- in accordance with 18 U.S.C. §1734 solely to indicate this fact. rent. 9971 Downloaded by guest on September 29, 2021 9972 Neurobiology: McGurk et al. Proc. Natl. Acad. Sci. USA 87 (1990) with a volume of approximately 0.3 ml. This chamber makes Spermine (1-1000 AM) alone did not produce any current in rapid changes in the bath solution possible. While recording, the oocyte, nor did it alter the membrane resistance of the oocytes were perfused with Mg2"-free amphibian Ringer's cell. The onset of the spermine effect was fast (T - 0.5 sec, solution (116 mM NaCl/2 mM KCl/1.8 mM CaCl2/10 mM Fig. 1D), which suggests that it was acting extracellularly and Hepes buffer, pH 7.2). NMDA, L-glutamate, and glycine not through uptake, a second messenger, or some other slow were obtained from Sigma. 7-Chlorokynurenic acid was signal transduction mechanism. The time constant of sper- kindly supplied by R. Keith (ICI Americas, Wilmington, DE). mine wash-off was slower than the onset (r - 1 sec; data not Spermine and putrescine were obtained from Aldrich. Except shown). Spermidine (500 ,.M) also potentiated the NMDA where indicated, test drugs were applied 10-15 sec before the response (data not shown). solution containing NMDA. If the glycine concentration was Potentiation of the NMDA-induced current by spermine not varied during the experiment, all solutions contained the increased with spermine concentration over the range 1-250 indicated concentration of glycine. Electrophysiological re- ,M (Fig. 2, * and A); at higher concentrations the effect cordings were carried out using a two-electrode voltage diminished (Fig. 2, o and A). Hill plots were fit to the rising clamp. The microelectrodes were filled with 1 M KCI (1-5 phase; that is, for spermine concentrations up to 250 ,M. The Mfl). Oocytes were clamped at a holding potential of -60 mV apparent Kd of the peak of the response was 39 ± 4 AM (Hill unless otherwise indicated. coefficient = 2.0 ± 0.1), while for the plateau response the Kd Concentration-response data were fit to the equation I = was 48 ± 6 ,uM (Hill coefficient = 1.9 + 0.2, n = 9). Imax[A/(A + Kd)I", where I is the response amplitude, and A Spermine could increase the NMDA response by a number is the concentration of the active agent. The maximum of mechanisms. It did not appear to alter the ioni selectivity current (Ymax), the dissociation constant (Kd), and the Hill of the channel. The current-voltage relationship of the coefficient (n) were fit as free parameters using a nonlinear NMDA response was linear between -60 and -20 mV both curve-fitting algorithm. All calculated values OfImax, Kd, and in the presence and in the absence of spermine, and the n are reported as mean + SEM. extrapolated reversal potential was unchanged (Fig. 3). Sper- RESULTS mine (250 ,M) also did not alter the Kd or Hill coefficient for The typical response to NMDA in the presence of low glycine NMDA (with 10 ,M glycine) but proportionally increased the is an early peak current that declines to a steady state or response at each concentration of NMDA, including saturat- plateau response as the receptor desensitizes (Fig. 1A, first ing levels (data not shown). record). Spermine reversibly potentiated both the peak and Spermine did affect the parameters of glycine action. the plateau phases ofthe NMDA response (Fig. 1A), but it did Concentration-response curves in which glycine concentra- not affect the short latency currents evoked by kainate or tion was varied with 200 ,uM spermine or no spermine (in the quisqualate or the long latency, inositol trisphosphate- presence of 250 ,M NMDA) indicated that the Kd as well as mediated response induced by quisqualate (Fig.