pl), and found three such domains in KCNQ2 and one in KCNQ3, containing five total potential NFAT-binding sites with the core motif GGAAA or TTTCC. Thus, we made four luciferase-reporter constructs encompassing the corresponding putative NFAT-binding domains, with luciferase expression as the readout for NFAT activation and binding MEK inhibitor to the reporter constructs ( Figure 6A). PC12 cells were transfected with the four luciferase-reporter constructs encompassing the corresponding putative NFAT-binding domains, and a constitutively active Renilla reniformis

luciferase construct. One day later, the cells were stimulated as before by regular Ringer’s, high K+, or ACh for 15 min, with termination by returning the cells to the culture medium. Cells were lysed after 2 days, and the resulting

luciferase luminescence was measured. Figure 6B shows the results from selleck KCNQ2 reporter constructs Q2RC1–Q2RC3 and the KCNQ3 reporter construct, Q3RC1. Significant firefly luciferase luminescence, normalized to the Renilla luciferase control, was observed 3 days after transfection for constructs Q2RC1–Q2RC3 and Q3RC1. Moreover, the luminescence increased at least 2-fold (p < 0.001) for constructs Q2RC1, Q2RC2, and Q3RC1, but not for construct Q2RC3, following stimulation of the cells by high K+ or by ACh (n = 5). There was a negligible response from cells transfected with empty vector for any stimulation. Our luciferase data predict regions Q2RC1 and Q2RC2 of the KCNQ2 gene and Q3RC1 of the KCNQ3 gene to be critical for transcriptional upregulation. Finally, exposure of cells to CsA for 1 hr before stimulation by high K+ or ACh did not alter the basal firefly luciferase luminescence for any of the reporter constructs;

however, the increased luciferase luminescence induced by high K+ or ACh was abrogated (n = 5) ( Figure 6C), suggesting that the reporter signals are due to CaN/NFAT. AKAP79/150 recruits CaN to multiple targets (Wong and Scott, 2004), including the CaV1.2 Ca2+ channel that serves as the Ca2+- and activity-dependent reporter that drives NFATc4 activation in the hippocampus (Oliveria et al., 2007). Thus, we probed the involvement of AKAP79/150 in CaN/NFAT regulation of M-channel expression in SCG neurons isolated from AKAP150+/+ (WT) and AKAP150−/− (KO) mice. We first transfected SCG neurons isolated from both groups of mice with EGFP-NFATc1 and NADPH-cytochrome-c2 reductase simultaneously monitored [Ca2+]i and EGFP-NFATc1 localization as previously described. We observed similar [Ca2+]i elevations for neurons isolated from both WT and KO mice (n = 14 and 20, respectively) but NFAT nuclear translocation only for neurons from WT mice (Figures 7A and 7B). Such data are summarized in Figures 7C and 7D (for statistics, see Supplemental Information). Thus, the absence of AKAP150 abolishes NFATc1 nuclear translocation induced by 50 K+ stimulation. We then compared IM levels between neurons isolated from AKAP150+/+ and AKAP150−/− mice by patch-clamp electrophysiology.