Figure 6.

Insertion Sites and Functional Screening of GluR1-GFP Fusion Proteins (A) A model of GluR1 topology showing the locations of the GFP insertion in 45 fluorescent fusion proteins. In-frame insertions of <TgPT-0> result in a 3 amino acid duplication (green) flanking the insertion site. In-frame insertions of <TcPT-1> generate only a 2 amino acid duplication (cyan) in the target because of the different reading frame. The orange amino acids are overlapping insertion sites of the two transposons (See supplemental diagram for the two reading frames). Multiple clones with identical transpositions are identified as 2x, 3x, etc. The six insertions resulting in functional, fluorescent GluR1-GFP/CFP tribrid fusion proteins are identified by the duplicated target amino acids (e.g. g209–211, c867–868). This figure was adapted from [43]. (B) AMPA receptor-mediated current from GluR1-CFP(867–868). (B1) Large whole-cell current elicited by the rapid sustained application of 5 mM glutamate (bar) in a cell transiently expressing GluR1-CFP(867–868) after reducing desensitization with cyclothiazide (100 μM). (B2) Current elicited by 5 mM glutamate in an outside-out patch pulled from a cell transiently expressing GluR1-CFP(867–868). (B3) Current elicited in the same patch as B3, but in the absence of cyclothiazide. Note the rapid and nearly complete desensitization of the current. (B4) The trace on the left is an expanded time scale of B3, the trace on the right is from an outside-out patch pulled from a cell transiently expressing wild-type GluR1 in the presence of 5 mM glutamate without cyclothiazide.