Analysis Of The Functional Differentiation Of The Neuronal NAChRs Gene Family In Vertebrates

Objective:Nicotinic acetylcholine receptors（nAChRs）are pentameric ligand-gated ion channels that play a crucial role in fast synaptic signaling in the central and peripheral nervous system.nAChRs are activated by the endogenous neurotransmitter acetylcholine and exogenous ligands such as nicotine.nAChRs are also closely related to a variety of neurological diseases such as myasthenia,schizophrenia,Parkinson’s disease,Alzheimer’s disease and nicotine addiction.Now,the molecular mechanism of nAChR subunits and formed receptors in the nervous system and their relationship with various diseases are not fully understood.With the rapid development of genomics and the accumulation of related data,it is possible to explore the functional differentiation of nAChRs gene family through evolutionary analysis.Here,via the phylogenetic analysis and maximum likelihood methods,we examined the evolution of the members of the nine vertebrate neuronal nAChR gene family.And we analyzed its evolutionary characteristics and the sites related to functional differentiation of subunits,as so to identify the receptor-specific sites.Methods:1.The homologous nucleotide sequences of protein coding region of the neuronal nAChRs gene family of twelve vertebrates were collected in this study.2.Processing the nucleotide sequence of all genes.Remove the nucleotides corresponding to the signal peptide fragments in each sequence,and all processed gene conduct multiple sequence alignment and construct phylogenetic tree.3.According to the phylogenetic tree,the selection pressure of each subunit and tribe,i.e dN/dS,was calculated by and codon replacement model and maximum likelihood method.4.Evolutionary rate of each subunit among vertebrates and among sites was calculated.5.Sites related to functional differentiation among subunits and among tribes of nAChRs are mapped to protein 3D structure,and analyze the relationship between structure and function.Results:1.Nine neuronal nAChRs subunits（α2-α7,β2-β4）were collected from twelve vertebrates by homology comparison,and 105 sequences were screened.2.Based on the translated amino acid sequences which are made on multiple sequences alignment of nucleotide sequences,observe a high degree of sequence identity in the allosteric coupling region,especially the M2-M3 loop,theβ1-β2 loop,the Cys-loop,theβ10-M1 helix junction,except theβ8-β9 loop.The highly conserved sites in the alignment were classified according to their presence in paralog or ortholog sequences separately before vertebrates.The conservation and variation of motifs in the ICD were also observed.3.The ML phylogenetic tree of nAChRs gene family shows the evolutionary relationship between subunits.They are clustered together by subtype.Topological relationships show that these subunits are more intimate in ortholog sequences than in paralog sequences.4.Each subunit and each tribe has different selection pressures relative to the rest.No positive selection was detected atα4,α7 andβ2 subunits.Positive selection was detected in other subunits,and a total of 60 positively selected sites were detected.The posterior probability of most sites was less than 0.95.Some of them accumulated in the N-terminal helix and others near theβ8 chain.Positive selection was detected in each tribe,and 54 sites of positive selection were detected,and the posterior probability was more than 0.95.The functional differentiation regions ofα2α4,α3α6 andβ2β4 tribes are in the ECD.In the tribe ofα5β3,most of positively selected sites were located in the TMDs.In theα3α6 tribes,many sites sit in theβ2chain including D-loop and theβ4-β5 loop.Theβ9 strand ofαsubunit of the heteropentameric receptor is the functional differentiation region.These positively selected sites can be used as the basis for studying functional differentiation.5.Each subunit has different evolutionary rates among sites and among vertebrates.In vertebrate,evolutionary analysis of each subunit showed that the ICD of each subunit was most divergent domain,and the change mode ofωvalues of ICD of each subunit was varied.Theωvalues of each site ofα7,α4 andβ2 subunits among species little change during the evolutionary process of vertebrates,excluding the ICD ofα4 andβ2 subunit,suggesting that the three subunits have strong functional or structural selective constraints.Theωvalues of TM4 of some subunits changed significantly,egα6,β3 andβ4 subunits,meaning that TM4 of these subunits showed functional or structural relaxed selective constraints.The event of positive selection was detected inα5 andβ2 subunits,and positively selected sites were located at N-terminal and ICD,respectively.It is necessary to explain that the ICD ofβ2 subunit has a remarkable evolutionary change.Conclusion:In vertebrates,the coupling regions of nine neuronal subunits are highly conserved,except forβ8-β9 loop.It shows that the importance of this coupling region for the signal transduction and positive selection seems to act on functional differentiation of subunits prior to the radiation of vertebrate orders.Furthermore,positively selected sites among subunits and among tribes favor functional specificity of subunits.These sites can be used as molecular basis for functional study.It is inferred that theβ9strand ofαsubunit of heteropentameric receptor contributes to agonists specificity,which is also related to the desensitization rate of nicotinic receptors.Theβ4-β5 loop is correlated with Ca²⁺permeability and allosteric modulation.In the process of evolution of vertebrates,α7,α4 andβ2 subunits possess a strong adaptability.Before vertebrates,their functions and structures evolved maturely.