These results indicate that the observed GABAAR phenotypes are not due to an intracellular transport defect caused by impaired NF transport. In addition, we performed immunocytochemistry

of the Kv3.1b channel in hippocampal neurons because KIF5s are involved in axonal transport of the Kv3.1b selleck products channel by direct binding (Xu et al., 2010). The distribution of Kv3.1b was indistinguishable between genotypes in both axons and dendrites (Figures S4A and S4B). Recently, KIF5s have been reported to interact with huntingtin-associated protein 1 (HAP1; known to be involved in GABAAR trafficking) via domains common to KIF5A, KIF5B, and KIF5C (Twelvetrees et al., 2010). However, among Kif5a-, Kif5b-, and Kif5c-KO mice ( Kanai et al., 2000; Tanaka et al., 1998; Xia et al., 2003), only Kif5a-KO mice show phenotypes related to an impairment of GABAAR trafficking in neurons. Kif5c-KO mice ( Kanai et al., 2000) and brain-specific Kif5b-KO mice (Y. Tanaka and N. Hirokawa, unpublished data) do not show epileptic seizure. These data suggest a specific role of KIF5A Verteporfin research buy in GABAAR transport, which cannot

be compensated by KIF5B or KIF5C. Thus, to gain an insight into the KIF5A-specific GABAAR-trafficking mechanism, we carried out yeast two-hybrid screening to identify proteins that interacted with KIF5A. KIF5A has 73 amino acids that have no homology with KIF5B or KIF5C ( Figure 4A). Using this region as bait, we identified a clone that encoded GABAAR-associated protein Isotretinoin (GABARAP) ( Wang et al., 1999) as a binding partner for KIF5A ( Figure 4B). Yeast two-hybrid experiments using deletion constructs of KIF5A, KIF5B, or KIF5C revealed that the C-terminal 73 amino acids of KIF5A were sufficient

for the interaction. KIF5B/KIF5C did not bind to GABARAP ( Figure 4B). Interactions were also detected between KIF5A and other GABARAP family members, namely GABARAP-L1 and GABARAP-L2 ( Figure 4C). The KIF5A-GABARAP interaction was further confirmed by a direct binding assay with purified recombinant proteins ( Figure 4D). Recombinant KIF5A showed an interaction with GABARAP, whereas recombinant KIF5B/KIF5C did not. The binding between KIF5A and GABARAP in vivo was further assessed by coimmunoprecipitation experiments using brain lysates (Figure 4E). Endogenous KIF5A was coimmunoprecipitated with endogenous GABARAP and GABAAR. Interestingly, HAP1 was not immunoprecipitated by an anti-GABARAP antibody (Figure 4E) but was coimmunoprecipitated with GABAAR (Figure 4F) as reported previously by Twelvetrees et al. (2010). GABARAP was not immunoprecipitated with an anti-HAP1 antibody (Figure 4G). These data suggest that the KIF5A-GABARAP complex is distinct from the KIF5-HAP1 complex (Twelvetrees et al., 2010). To examine the relationship of KIF5A with GABARAP, we studied the subcellular localization of KIF5A and GABARAP in cortical neurons by immunocytochemistry.