ASN-265 OF FROG KAINATE BINDING-PROTEIN IS A FUNCTIONAL GLYCOSYLATIONSITE - IMPLICATIONS FOR THE TRANSMEMBRANE TOPOLOGY OF GLUTAMATE RECEPTORS

Kainate binding proteins (KBPs) from frog acid goldfish brain are glyc osylated, integral membrane proteins. These KBPs are homologous (35-40 %) to the C-terminal half of AMPA and kainate receptors which have bee n shown to form glutamate-gated ion channels. We report here that the frog KBP has three functional N-glycosylation sites, Of particular int erest, Asn-265, a residue located between two putative membrane spanni ng regions of the frog KBP, is a functional N-glycosylation site. A mu tation of Ser-267 to Gly renders this site non-functional as shown usi ng an in vitro translation system and by transient expression in human embryonic kidney (HEK 293) cells. The mutant receptor protein (S267G) , when expressed in HEK cells, binds kainate with high affinity (K-d = 16 nM). These results further support a topology with three transmemb rane segments for KBPs and, by sequence homology, for glutamate-gated ion channels.