Divalent Ion Permeability of AMPA Receptor Channels Is Dominated bv the Edited Form

N. Burnashev,* H. Monyer,+ P. H. Seeburg,+ and B. Sakmann* *Max-Planck-lnstitut fur medizinische Forschung Abteilung Zellphysiologie Heidelberg Germany +Center for Molecular Biology Laboratory of Molecular Neuroendocrinology University of Heidelberg Germany Summary Functionally diverse GluR channels of the AMPA sub- type are generated by the assembly of CIuR-A, -B, -C, and -D subunits into home- and heteromeric channels. The CIuR-B subunit is dominant in determining func- tional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and un- edited forms, GIuR-B(R) and CluR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GIuR-B(R) channels expressed in 293’ cells display a low divalent permeability, whereas homo- merit CIuR-B(Q) and GIuR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine and glutaminecontaining subunits generates cells with varying divalent permeabilities de- pending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GIuR-B(R) subunit determines low divalent permeability in heteromeric CluR channels and that changes in CIuR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell. Introduction Glutamate-operated ionchannels(GluRchannels)dis- play diverse functional properties. The shape of their current-voltage (I-V) curves and their divalent perme- ability properties, measured with glutamate-activated whole-cell currents, depend on the cell type exam- ined (Mayer and Westbrook, 1987; Ascher and Nowak, 1988; lino et al., 1990; Gilbertson et al., 1990; Huettner, 1990; Ozawa et al., 1991). Comparison of functional properties of recombinant AMPA receptor channels formed from GIuR-A, -B, -C, and-D subunits and their combinations (Hollmann et al., 1989; Keinlnen et al., 1990; Boulter et al., 1990; Nakanishi et al., 1990; Saki- mura et al., 1990; Sommer et al., 1990; Verdoorn et al., 1991) with those of native CluR channels in rat hippocampal slices suggested that theGIuR-B subunit is dominant in determining functional properties of native GluR channels of the AMPA subtype (Jonas and Sakmann, 1991). Mutational analysis identified a sin- gle amino acid difference (arginine versus glutamine) in a particular position of the putative channel-form- ing segment TM2 (Keinlnen et al., 1990) as the molecu- lar determinant of subunit-specific differences in channel conductance (Verdoorn et al., 1991). In this position (Q/R site), the GluR-B subunit carries an argi- nine, whereas the GIuR-A, -C, and -D subunits contain a glutamine residue. Expression studies of hetero- merit GluR channels in Xenopus laevis oocy-tes indi- cated that the divalent permeability of recombinant channels is governed by the presence or absence of the GIuR-B subunit (Hollmann et al., 1991) and that this property is also dependent on the GluR-B-specific arginine residue in TM2 (Hume et al., 1991). It was recently found, however, that the GluR-B gene speci- fies a glutamine residue at the Q/R site of TM2 and suggested that the arginine in this functionally critical position is introduced by GIuR-B mRNA editing (Som- mer et al., 1991). This editing process appears to be highly selective, as no arginine-containing TM2 oc- curs in the GIuR-A, -C, and -D channel subunits of adult animals. We now report evidence suggesting that GIuR-B mRNA editing is developmentally controlled and that the unedited GIuR-B(Q) subunit coexists with GIuR- B(R) in fetal brain. To understand the molecular basis of the “dominance” of the edited GIuR-B subunit (GIuR-B(R)) in determining the properties of native AMPA receptor channels, we characterized the diva- lent permeabilities of recombinant channels assem- bled from the two TM2 forms of GIuR-8’ and from the GluR-D subunit, using a mammalian expression system. The data indicate that the edited form of GIuR-B subunits controls the low divalent permeabil- ity of heteromeric assemblies as a result of the func- tional dominance of the arginine residue in its chan- nel-forming TM2 segment. Heteromeric channels may coexist in the same cell with homomeric channels with high divalent permeability, suggesting that the glutamate-activated divalent permeability of single cells may represent the superposition of currents me- diated by a mosaic of both divalent impermeable het- eromeric and divalent permeable homomeric AMPA receptor channels. Results Edited and Unedited Versions of the CIuR-B Subunit In heteromeric assemblies of AMPA receptor sub- units, GIuR-B determines the properties of monova- lent cation flow by virtue of the unique arginine resi- due in its TM2 region. This particular arginine is not encoded on the GIuR-B gene, but seems to be intro-