Figure 8.
AKAP signaling complex facilitates BK-induced phosphorylation of IP3R1 and the sensitization process. (A) Lysates of PC12 cells transfected with either AKAP-IS or the scrambled sequence were immunoprecipitated with an antibody against the regulatory subunit (RIIα) of PKA, and precipitates were analyzed by blotting with the indicated antibodies. (B) Anchored pool of PKA is required for BK-induced phosphorylation of IP3R1. PC12 cells transfected with either AKAP-IS or the scrambled sequence were treated with vehicle or BK, and PKA-dependent phosphorylation of IP3R1 was examined as in Fig. 7. (C) Sensitization is facilitated by the AKAP signaling complex. PC12 cells transfected with either AKAP-IS or the scrambled sequence were loaded with fura-2 for [Ca2+]i measurements. (D) Involvement of yotiao in the sensitization process. (Top) Expression of yotiao mRNA in the control and yotiao siRNA-transfected cells was analyzed by RT-PCR as described in Materials and methods. Results from RT-PCR performed with β-actin primers are presented as a control to verify that equal amount of cDNA was used in each reaction. Inverted imaged are shown. (Bottom) Control and yotiao siRNA-transfected cells were loaded with fura-2 for [Ca2+]i measurements. (C and D) Cells were stimulated with BK (1 μM, 5 min) followed by EGF (20 nM), and the peak heights in the BK- and EGF-induced [Ca2+]i increase were measured. Error bars are mean ± SEM of three experiments. *, P < 0.05; **, P < 0.01.