Furthermore, knockdown of TRIP8b in vivo

resulted in an increased immunoreactivity for HCN1 channels in the CA1 soma and proximal dendrites that represents a redistribution of HCN1 to intracellular compartments. Additionally, coexpression of EGFP-HCN1 with TRIP8b siRNA revealed a selective loss of channel fluorescence in SLM. All together, these results indicate that, in addition to being important for HCN1 expression on the plasma membrane, TRIP8b may 3-MA cell line also be important for the targeting of HCN1 to distal dendrites. However, the loss of HCN1 in distal dendrites might not reflect a specific role of TRIP8b in dendritic targeting but may be secondary to the general loss of HCN1 surface expression upon TRIP8b knockdown. Moreover, because the TRIP8b siRNA reduced but did not eliminate TRIP8b protein, it is unclear whether the residual targeting of HCN1 to the distal dendrites results from an effect of residual TRIP8b or represents the action of some other targeting protein that interacts with HCN1. To address these questions, we adopted a third, complementary approach, discussed next. To overcome the limitations of the siRNA approach, we expressed an EGFP-tagged BMN 673 datasheet HCN1 truncation mutant (EGFP-HCN1ΔSNL) that lacks the HCN1 C-terminal SNL tripeptide required for high affinity binding of HCN1 to TRIP8b (Santoro et al., 2004, Santoro et al., 2011 and Lewis

et al., 2009). We observed a dramatic loss of dendritic targeting when we expressed EGFP-HCN1ΔSNL in the background of HCN1 KO mice (Figures 4A and 4B). Unlike wild-type HCN1, the mutant channel was expressed uniformly at high levels throughout CA1, as evident in the relatively constant EGFP-HCN1ΔSNL to DsRed2 fluorescence ratio along the somatodendritic axis. A comparison with the distribution of full-length HCN1 revealed second not only a loss of expression of the mutant channel in the distal dendrites but also an increase in expression in proximal dendrites (Figures 4C and 4D; EGF-HCN1: N = 4 mice, 8 injection sites;

EGFP-HCN1ΔSNL: N = 5 mice, 10 injection sites). As TRIP8b is the major protein that interacts with the HCN C terminus in the brain (Santoro et al., 2004, Santoro et al., 2009 and Zolles et al., 2009), these results strongly implicate TRIP8b as a key element necessary for the efficient targeting of HCN1 channels to distal portions of CA1 pyramidal neuron apical dendrites. Because of the limitations of fluorescence imaging, we used an electrophysiological approach to measure EGFP-HCN1ΔSNL channel levels in the surface membrane in HCN1 KO mice. The resting potential of neurons expressing EGFP-HCN1ΔSNL (−69.2 ± 1.2; n = 13) was identical to that of neurons expressing EGFP-HCN1 (−69.1 ± 1.1 mV; n = 15), and both were ∼14 mV more positive than the resting potential of control neurons from the HCN1 knockout mice expressing EGFP (−82.7 ± 1.5 mV; n = 15; p < 0.