26

3 A PURIFIED FUNCTIONAL NON-NMDA GLUTAMATE RECEPT(.)R RECONSTITUTED IN ARTIFICIAL LIPID BILAYERS. A. Ambrosini, G. Prestipino*, J.M. Henley and E.A.Barnard. MRC Molecular Neurobiology Unit, MRC Centre, CB2 2QH, Cambridge, U.K., and *Istituto di Cibernetica e Biofisica, CNR,I-16146, Genova , Italy.

Xeno~us central nervous system provides an abundant source of kainate and quisqualate ionotropic receptors. [3H]-

kainate (KA) and [3H]-AMPA (a quisqualate derivative) binding sites have been efficiently solubilised from this tissue

by the detergent n-octyl-13-glucoside. In these detergent extracts, [3H]-KA and [3H]-AMPA bind with Kd = 20 and 170 nM respectively. Affinity chromatography with immobilised domoic acid (DA), a potent KA agonist (Hampson D.R. and Wenthold R.L-1988) has been employed to purify the KA binding sites, with protection from proteases. Several bands were consistently identified on SDS gel electrophoresis of th,." purified preparation: a major component at Mr = 42 KDa and other bands at Mr = 48, 52, and 55 KDa. The affinit) purification resulted in a concomitant enrichment in AMPA sites. The KA and AMPA binding sites presented similar pharmacological properties for both the solubilised extract and the purified preparation, the rank order of potency of competitive ligands in inhibiting binding being DA>KA>AMPA>Glu=CNQX for both sites. The purified KA and AMPA sites were reconstituted in a:tificial lipid vesicles. Subsequently, they were incorporated into planar lipid bilayers and agonist-dependent currents and single channels were recorded. Single channels were resolved that were specifically activated by glutamate agonists. Their properties show that the purified complex represents a functional non-NMDA glutamate receptor.

RELATION BETWEEN PIGEON AND XENOPUSKAINATE RECE.Pq'ORS: AN IMMUNOLOGICAL STUDY. A. Ambrosini, P. Streit* and E.A.Barnard. MRC Molecular Neur~biology Unit, MRC Centre, CB2 2QH, Cambridge, U.K., and *Brain Research Inst., Univ. of Zurich , CH.

A monoclonal antibody (mAb) was developed which precipita, ed kainate (KA) binding activity from detergent extract of pigeon cerebellum. This mAb allowed to isolate a KA binding protein by immunoaffinity chromatography (Klein A.U. et al., 1988), which migrated in SDS PAGE at Mr = 50 KDa. Fttrther, a polyclonal antibody (pAb) was produced, which was able to recognise in western blotting analysis this pigeon KA binding protein. Now, we present evidence that these antibodies cross-react witt: a KA binding protein from Xenapus CNS. In XenopusCNS,

[3H]-KA and [3H]-AMPA (a potent quisqualate analogue) bind in a competitive fashion to the same protein complex (a putative non-NMDA receptor) and can be purified in a 1:1 ratio by domoic acid affinity chromatography (see also the

abstract by Ambrosini A. et al). The mAb developed against pigeon KA binding protein immunoprecipitated both [3H]- KA and [3H]-AMPA binding activities from Xenoous solubili~ed extract and a purified KA/AMPA binding protein. In addition, the pAb labelled a band at an apparent Mr of 42 KDa, in immunoblotting experiments performed on Xenot~us solubilised extract and purified material. This band correspo:lds to the major component of the non-NMDA receptor complex affinity-purified from XenopusCNS.

5

EXCITATORY AMINO ACID-INDUCED CHANGES IN EXTRA-CELLULAR CALCIUM CONCENTRATION IN THE RAT HIPPOCAMPUS INDICATE DIFFERENT ACTIVATION OF THE NA +/CA 2 + EXCHANGER Y. Arens, Y. Stabel & [7.. Heinemann, Inst. Neurophysiologie, Univ. KOln, Robert-Koch-S~.39, 5000 K~ln41, FRG In the CNS all excitatory amino acids produce dose-dependent decreases in [Ca~T]o and [NaT]o . Hqwever the specific agonists quisqualate, kainate and AMPA induce in addition to the initial decrease a subsequent increase of [Ca 2 + ]o above baseline which outlasts the application time. NMDA, DL-homocyst~ic acid and the mixed agonists glutamate and aspartate do not produce an

2 + 2 + overshooling [Ca ]o response. The direction of Ca -fluxes across the eell membrane and therefore also the activation of the + z + 4 , 2+ Na /Ca exchanger is determined by the electrochemical Na - a n d Ca -gradients and the transmetnbrane potential. To test the

+ 2+ 2+ hypothesis that activation of the Na /Ca exchange mechanism is involved in the produaion of the Ca -overshoots we studied the effects of low [Na+ ],, low [K+]~, Ouabain (5 uM) and lithium (5 raM) on quisqualate-induced [Ca2+]o changes. All these treatments enhanced the jCa 2 +'1o decreases" and reduced the overshooting responses in a reversible manner. In some cases lithium could totally block the [Ca z + ]o-OVershoots which may be explained by a direct blocking effect of lithium on the action of the Na + ~Ca 2 + exchanger. To clarify further the basic mechanisms which are involved in the generation of quisqualate-induced Ca 2 + -overshoots we tested the effect of dantrolene, ryanodlne and caffeine; drugs which may influence the rgJcaae of intracellular calcium. Dantrolene (20 u_M) and ryanodine (20 uM) had no effect, whereas caffeine (5 raM) reduced the [Ca2+]o decreases but d idno t affect the overshoots. The

,_24- 2 + 2+ reduction of [Ca ]o decreases by caffeine may be explained by a Ca -dependent inactivation of Ca currents due to an enhanced 2+ , 2 + intracellular [Ca ]. Whatever mechanism is re~a~onsible for the generation of the [Ca ] overshoots, they may be of fundamental

importance for the maintenance of intracellular Ca 2 + homeostasis,