EFFECT OF RNA EDITING AND SUBUNIT COASSEMBLY ON SINGLE-CHANNEL PROPERTIES OF RECOMBINANT KAINATE RECEPTORS

1. Patch-clamp methods have been used to examine single-channel proper ties of recombinant GluR5 and GluR6 kainate-preferring glutamate recep tors which differ in a single amino acid residue as a result of RNA ed iting at the Q/R (glutamine/arginine) site. Subunits were expressed al one or in combination with the high-affinity kainate receptor subunit KA-2 in transfected human embryonic kidney (HEK-293) cells. 2. In outs ide-out patches, unedited homomeric GluR6(Q) receptors exhibited direc tly resolved domoate-activated single-channel conductances of 8, 15 an d 25 pS. Variance analysis of GluR6(Q) responses gave a mean conductan ce of 5.4 pS, while the edited isoform GluR6(R) had an unusually low c hannel conductance (225 fS). 3. Homomeric channels composed of GluR5(a ) subunits exhibited. three conductance states of 5, 9 and 14 pS chara cterized by prolonged burst activations in the presence of domoate. In contrast, the GluR5(R) subunit, which has not previously been reporte d to form functional homomeric receptors, had an extremely low conduct ance (< 200 fS). 4. Heteromeric GluR6(Q)/KA-2 kainate receptors gave s ingle-channel events indistinguishable from homomeric GluR6(Q) channel s. Conversely, openings produced by GluR5(Q)/KA-2 and GluR5(a) recepto rs differed from each other in their kinetic properties. The primary e ffect of co-expression of KA-2 with GluR5(a) was a dramatic shortening in channel burst length. 5. Spectral and variance analyses were used to estimate mean single-channel conductances of heteromeric edited rec eptor-channels; channel conductances were 950 fS for GluR5(R)/KA-2 rec eptors and 700 fS for GluR6(R)/KA-2 receptors. Both receptor types had significantly higher conductances than the respective homomeric chann els, GluR5(R) and GluR6(R). 6. We conclude that Q/R site editing drama tically reduces single-channel conductance. Furthermore, we find simil arity between the kainate receptor-channels described in sensory neuro nes and the recombinant GluR5(a) homomeric channel. Characterization o f recombinant single-channel properties could therefore aid identifica tion of the native kainate receptors.