A UNITARY NON-NMDA RECEPTOR SHORT SUBUNIT FROM XENOPUS - DNA CLONING AND EXPRESSION

A high-affinity homomeric, non-NMDA glutamate receptor was previously purified from the amphibian Xenopus laevis. We have obtained nine pept ide sequences from its submit, applied in cDNA cloning. The cDNA encod es a subunit (XenU1) containing all nine sequences. The 51,600-dalton mature subunit has four hydrophobic domains homologous to the four in the C-terminal half of mammalian non-NMDA receptor submits. Transient expression in COS cells showed 1:1 binding (at B-max) of [H-3] kainate (K-D = 9.1 nM) and of [H-3] AMPA (alpha-amino-3-hydroxy-5-methyl-isox azole-4 acid; K-D = 62 nM). The competitive binding series domoate > k ainate > AMPA > NBQX > glutamate was established (where NBQX is 2,3-di hydroxy-6-nitro-7-sulphamoyl-benzo (f) quinoxaline). Each agonist show s the same K-I value against [H-3] kainate and [H-3] AMPA binding, sug gesting a common agonist site, but two conformations thereof are disti nguishable by their different affinities for the antagonist NBQX and b y the allosteric effect of thiocyanate anion (greatly potentiating AMP A binding, inert with kainate). XenU1 is exceptional among non-NMDA re ceptor submits because it lacks most of the large N-terminal domain fo und in those of mammals and it has high affinity for both kainate and AMPA. It differs from the similarly-short ''kainate-binding proteins'' (KBPs), in binding AMPA and in forming glutamate receptor channels wh en the native protein is reconstituted. Moreover, whereas a full-lengt h kainate receptor of mammals, GluR6, is shown here (from a partial cD NA sequence) to exist also in Xenopus, with similar to 97% sequence id entity to rat GluR6, XenU1 is much less homologous to any rat kainate or AMPA receptor and also to the KBPs, even from another amphibian, Ra na. Another difference is that a potential concensus sequence (''EF ha nd'') for Ca2+ binding is present in the N-terminal domain of XenU1, b ut not in the chicken (glial) KBP. XenU1 is deduced to be in a new fam ily of non-NMDA receptors.