There was no statistical difference in IM amplitudes from the two groups of neurons when treated with Ringer’s solution (0.87 ± 0.05, n = 18, and 0.83 ± 0.09, n = 20), suggesting that the lack of AKAP150 does not affect basal expression levels of M channels. However, the effect of augmented IM amplitudes after 50 K+ treatment (1.28 ± 0.08 pA/pF, n = 38; p < 0.01) was wholly absent in neurons from AKAP150−/− mice (0.81 ± 0.08 pA/pF, n = 28) ( Figures 8A and 8B). To test whether the aforementioned result in AKAP150 KO neurons is truly due to the lack

of AKAP150, such neurons were transfected with WT AKAP79, AKAP79 with a PKC-binding domain deletion (ΔA-AKAP79) (Klauck et al., 1996; Zhang et al., 2011), or AKAP79 with a CaN-binding domain deletion (AKAP79ΔCaN) (Klauck et al., 1996; Oliveria et al., 2007) to “rescue” the responses of augmented expression of M channels after high-K+ stimulation. Transfection of EGFP-tagged AKAP79 (n = 9) or ABT-263 selleck kinase inhibitor ΔA-AKAP79 (n = 16), but not AKAP79ΔCaN (n = 9), in the AKAP150−/− neurons restored the augmented IM amplitudes after 50 K+ stimulation, suggesting the role of AKAP79/150 and its recruited CaN, but not PKC, in NFAT-mediated regulation of M-channel expression ( Figure 8C). Within the CaN-binding site on AKAP79/150 is the sequence, PIAIIIT, satisfying the consensus CaN-binding sequence PIXIXIT, and its deletion prevents the CaN-AKAP79/150

interaction ( Oliveria et al., 2007). This deletion mutant (AKAP79ΔPIX) also failed to rescue the responses of augmented IM amplitudes after 50 K+ stimulation in AKAP150 KO neurons (n = 9) ( Figures 8C and 8D; for statistics, see Supplemental Information). We noticed that the “rescued” upregulation of IM in AKAP150−/− neurons transfected with AKAP79 or ΔA-AKAP79 was significantly larger than those in neurons from AKAP150+/+ mice (compare Figures 8B and 8D; p < 0.01), probably because AKAP79 was overexpressed in these neurons. However, such AKAP79 overexpression did not upregulate IM amplitudes in neurons treated with regular

Ringer’s solution, much further confirming the role of AKAP79/150 and CaN-mediated NFAT signaling in activity-dependent, but not tonic, transcriptional regulation of M channels. To determine the source of Ca2+i signal that activates the CaN/NFAT, we tested the effect of bradykinin (BK) receptor stimulation, nominally Ca2+-free external solution, or various subtype-specific Ca2+-channel blockers on both NFAT nuclear translocation and upregulation of IM amplitude. We first explored whether NFAT activation requires influx of Ca2+ through the plasma membrane, using imaging on SCG neurons from WT mice transfected with EGFP-NFATc1. Neurons loaded with fura-2 were stimulated by either BK (250 nM), which stimulates Gq/11-coupled B2 receptors to induce Ca2+ release from IP3-sensitive Ca2+i stores (Cruzblanca et al., 1998; Gamper and Shapiro, 2003; Zaika et al.