Figure 1. Characterization of PKA-anchoring deficient AKAP150ΔPKA mice.

(A) Diagram depicting the mouse Akap5 gene encoding the AKAP150 WT allele (top), the targeting construct containing the ΔPKA mutation (middle), and the targeted ΔPKA allele (bottom). The single AKAP150 coding exon is represented by the black box. The red rectangle indicates the 30 bp encoding the 10 amino acids of the ΔPKA deletion, yellow rectangle indicates the in-frame insertion of a c-myc epitope tag at the AKAP150 C-terminus, and green triangles indicate loxP sites flanking the neomycin resistance cassette in the 3′ genomic DNA. (B) Diagram of AKAP150 protein primary structure indicating removal of 709-LLIETASSLV-718 to selectively disrupt PKA-RII anchoring. (C) PCR-based genotyping of WT and heterozygous and homozygous AKAP150ΔPKA littermate mice. (D) Detection of AKAP150ΔPKA protein in whole-cell hippocampal extracts from homozygous mice by anti-myc and anti-AKAP150 immunoblotting (IB). (E) The AKAP150ΔPKA mutation or AKAP150 KO (−/−) eliminates anti-AKAP150 co-immunoprecipitation (IP) of PKA-RII and C subunits but not CaNA. Ext = whole-cell hippocampal extract. IgG = IP with non-immune immunoglobulin. (F) HEK293 cells co-transfected with AKAP79 WT or ΔPKA-CFP (magenta) and PKA RII-YFP subunits (green). Co-localization appears white in the merged panel. (G and H) tsA-201 cells co-transfected with C or N-teminally tagged Ca_V1.2-YFP (FRET acceptor, green) and WT or ΔPKA AKAP79-CFP (FRET donor, blue). Corrected FRET (FRETc) shown in pseudocolor gated to CFP. (I and J) Quantification of apparent FRET efficiency measured between Ca_V1.2-YFP and WT or ΔPKA AKAP79-CFP. Data expressed as mean ± SEM. (Not significantly different by t-test; n = 7–17). Scale bars = 10μm.