| Objective:Nicotinic acetylcholine receptors(nAChRs), ligand gated ion channel pentamers made up of five subunits, are widely expressed in both the central nervous system(CNS) and the peripheral nervous system(PNS). As the major receptors of the neurotransmitter acetylcholine(ACh), they play important roles in neuronal signaling transduction. They are also the receptors activated by nicotine in tobacco smoking and thus are the key molecules in the development of smoking addiction. Moreover, abnormalities in nAChRs have also been demonstrated to be related to various nervous system diseases. So far, there was no research associated nAChR subunits with functional divergence. In this study, we conducted a phylogenetic analysis of nAChR subunits in vertebrates to investigate their molecular evolution features. Furthermore, we analyzed functional divergence and related sites in the neuronal-type subunits. Methods:1. The protein sequences of nAChRs subunits of eight vertebrates were collected, i.e., Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus tropicalis and Danio rerio. Based on the sequences, two data sets were crurated, i.e., one data set contained all the collected sequences, and the other included only the sequences of neuronal-type subunits.2. We processed those collected sequences by removing the redundant sequences firstly and deleting the signal peptides manually. Then we conducted multiple alignments and phylogenetic analysis of these nAChR subunits.3. We predicted the physicochemical property of α7 subunits and showed the key information on multiple alignments.4. We conducted multiple alignments and phylogenetic analysis of neuronal nAChR subunits, and calculated the functional divergence coefficients among those subunits..5. We conducted a sites-special profile to infer the functional divergence related sites and chose those sites under the condition Qk>0.67. Results:1. Totally, we collected 122 sequences of 16 subunits, i.e., α1-α7, α9, α10, β1-β4, γ, δ and ε. The data set consisting of the neuronal-type subunits included 9 members, i.e., α2-α7、β2-β4, and 69 sequences were included.2. The phylogenetic tree showed the evolutional relation among different subunits. The subunits β1, γ, δ and ε formed one cluster, which belonged to the muscle type. The subunits α1-α6, β2-β4 formed one cluster, which belonged to the neuronal type except the α1. And subunits α7, α9 and α10 formed one cluster, which belonged to the third type. The close subunits such as β2 and β4, α2 and α4, α6 and α3, α5 and β3 also had similar function.3. The secondary structure of α7 showed the conserved α-helix or β-fold. The β-folds formed the loops in binding domain and in each transmembrane segment there is α-helix.4. There were three divergence events among neuronal subunits(α2-α7, β2-β4). The divergence I diverged homogeneous subunits α7 and other heterogeneous subunits(α2-α6, β2-β4). The divergence II diverged α-type subunits and β-type subunits. The divergence III happened among α-type subunits. The functional divergence coefficient θ >0(p-values<0.05) meant there may be a divergence, and the values of θ reflected the probability. Furthermore, the values of θI reflected the site-specific evolutionary rate shift, resulting in the divergence among α-type subunits. The values of θII reflected the site-specific evolutionary property shift, resulting in the divergence among α-type subunits and β-type subunits.5. Pair-wise comparisons between heterogeneous subunits(β4, β2, α4, α2, β3, α5, α6, α3) and homogeneous subunits(α7) were conducted respectively. There were 261 functional divergence related sites in total. Under the condition(Qk>0.67), we got 16 these sites could be molecular basis for related research. |
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