05 ANOVA with Tukey’s HSD, 25 and 30 V stimulus strengths). These results demonstrate that while full-length HCN1 is targeted to CA1 distal dendrites, the truncation mutant is expressed at high, relatively uniform levels in the somatodendritic membrane throughout the CA1 neuron, consistent with our results based on EGFP fluorescence. Thus, the loss of distal dendritic targeting

of HCN1ΔSNL is not secondary to loss of membrane surface expression but must represent the loss of a primary action of TRIP8b to target full-length HCN1 to distal dendrites. As downregulation of TRIP8b with siRNA decreases HCN1 surface expression, the HCN1ΔSNL results further indicate that the actions of TRIP8b to enable proper surface membrane expression and to direct distal dendritic targeting of HCN1

are dissociable functions. This is consistent with recent reports that HCN1 and TRIP8b interact click here at two distinct sites ( Lewis et al., 2009 and Santoro et al., 2011) and that the weakened binding between TRIP8b and HCN1ΔSNL is sufficient to allow certain TRIP8b isoforms to enhance surface expression of the mutant channel (see Discussion). Although our results buy ERK inhibitor indicate that TRIP8b is critical for the proper surface expression and dendritic targeting of HCN1 in CA1 pyramidal neurons, these data do not provide insight as to which specific TRIP8b isoform (or combination of isoforms) is involved. The identification of the role of individual isoforms is a daunting task as there are at least ten different Casein kinase 1 splice variants

of TRIP8b expressed in brain (Lewis et al., 2009 and Santoro et al., 2009). Moreover, the small size of the various alternatively spliced exons makes it impractical to design selective siRNAs to knockdown specific isoforms. Nonetheless, we obtained insight into the function of specific isoforms by examining a mouse line, Pex5ltm1(KOMP)Vlcg, in which exons 1b and 2 in the TRIP8b gene were replaced by lacZ through homologous recombination (http://www.komp.org; Figure S2). The removal of all splice forms containing exons 1b or 2 is expected to delete all except three of the TRIP8b splice isoforms, namely TRIP8b(1a), TRIP8b(1a-4) and TRIP8b(1a-3-4). Of these, TRIP8b(1a) and TRIP8b(1a-4) are the most abundant splice forms in the mouse brain, accounting for 25%–30% and 30%–40%, respectively, of total TRIP8b mRNA. In contrast, TRIP8b(1a-3-4) is normally expressed at very low levels in brain (<5% of total brain TRIP8b mRNA; (Santoro et al., 2009) and is not detected in hippocampus (Lewis et al., 2009). The TRIP8b exon 1b/2 KO mice are generally viable, with normal body weight and overall brain structure. Western blot analysis of brain extracts from these mice confirmed the loss of all TRIP8b isoforms containing exons 1b or 2.