Figure 1. CaV1.2 and AKAP79/150 interact directly.
(A) Lysates from primary cultured hippocampal neurons (12–17 DIV) were immunoprecipitated (IP) and then immunoblotted (IB) with anti-AKAP150 or anti-CaV1.2 C-terminal (CT4) antibodies (n = 3). Extract lane exposure times were 5 min, all other exposure times were 1 min. Control lane (IgG, right), IP with non-specific rabbit IgG.
(B) Top, Alignment of human AKAP79, rat AKAP150, and orthologs from other species showing conservation of heptad-spaced hydrophobic residues between the end of the PKA-RII anchoring region and the C-terminus. Bottom, LZ motif alanine substitution mutants (LZm) introduced into AKAP79 and CaV1.2.
(C) Micrographs of transfected HEK293 cells showing subcellular localization of AKAP79-CFP (donor), CaV1.2-YFP (acceptor), CFP-YFP colocalization (Merge), and corrected, sensitized CFP-YFP FRET (FRETc; monochrome), and FRETC gated to donor intensity (FRETC(CFP); pseudocolor). N and C indicate fluorophore fusion to the N- or C-terminus.
(D) Effective FRET efficiency (Eeff) calculated from both 3-filter set measurements (3F) and YFP-acceptor photobleach (PB) measurements from the same cells (n values indicated in parentheses; * P = 0.025, # P = 0.018, *** P = 2.46 × 10−6, ### P = 4.20 × 10−7, relative to the upper row of data).
(E) Left, LZ mutations disrupt coIP of CaV1.2 and AKAP79 in HEK 293 cells. Lysates from cells transfected with either wt or LZm forms of CaV1.2 and AKAP79-CFP were IP’d with the anti-CaV1.2 CT4 antibody and then probed by western blotting with an anti-GFP antibody that recognizes CFP-fused AKAP79. The same blot was stripped of anti-GFP and re-probed with the anti-CaV1.2 CT4 antibody. Extract lane was loaded with 50 μg total protein, CaV1.2 band is visible on the strip and re-probe blot at longer exposure times as in A. Right, Western blot of 40 μl IP input lysates. Untransfected control lysate marked as untx.