To locate the S435 (β4) and D397 (α5) residues within the receptor pentamer, we performed homology modeling with the Torpedo nAChR using one possible α3β4α5 subunit arrangement. This

model predicted the formation of a very similar disposition of α helices in the α3β4α5 and mapped both residues to the intracellular vestibule ( Figure 2B). Electrostatic mapping of the vestibule showed a particular disposition of charges with S435 and D397 located at the more distal and positively charged part of the vestibule ( Figure 2C). These data indicate first that the critical residue in β4 that mediates the β4 effect is located in the receptor structure near the most common SNP of α5 to be associated with heavy smoking; and second, that this is a highly charged domain of the receptor where single residue changes may have a particularly strong effect on receptor activity. To test the hypothesis Protease Inhibitor Library manufacturer that β4 is rate limiting for nAChR Akt inhibitor assembly and function in vivo and that overexpression of β4 can strongly influence nicotine-evoked currents and behavioral responses to nicotine, we characterized a bacterial artificial chromosome (BAC) transgenic line spanning the Chrnb4-Chrna3-Chrna5

gene cluster ( Gong et al., 2003). The BAC transgene included the intact coding sequences of the Chrnb4 gene, modified sequences of Chrna3, and incomplete sequences of Chrna5. Chrna3 was modified by insertion of an eGFP cassette followed by polyadenylation signals at the ATG translation initiator codon of Chrna3 ( Figure 3A). The upstream sequences of Chrna5, encoding exon 1 splice variants ( Flora et al., 2000), are missing in the BAC transgene ( Figure 3A). To promote correct expression of Chrnb4, the BAC included the intergenic and 5′ flanking regions encompassing the cis-regulatory elements that coordinate cotranscriptional control of the genes in the cluster ( Bigger et al., 1997, Medel and Gardner,

2007 and Xu et al., 2006). As a result of these modifications in the BAC transgene, these Tabac mice express high levels of β4, but not α5 ( Figure 3B), and expression of α3 is replaced by expression of an eGFP reporter cassette to monitor the sites expressing the transgene ( Figures 3C–3H). As shown in Figure 3, neurons science expressing eGFP were evident in autonomic ganglia ( Figure 3C), and in very restricted brain structures ( Figures 3D–3H) known to express these genes ( Zoli et al., 1995). Immunostaining with cholinergic (ChAT) and dopaminergic (TH) markers indicated high expression of Chrna3/eGFP in ChAT neurons of the Hb-IPN system ( Figures 3G and 3H). Intense Chrna3/eGFP expression was also detected in other brain areas ( Figures 3D and 3E) involved in nicotine addiction, such as the ventral tegmental area (VTA), the caudal linear nucleus (Cli), the supramammilary nucleus (SuM) ( Ikemoto et al., 2006), and the laterodorsal tegmental nucleus ( Figure 3F), which provides modulatory input to the VTA ( Maskos, 2008).