Structural And Functional Studies Of The Drosophila Transient Receptor Potential Ion Channel Pyrexia

Transient receptor potential ankyrin(TRPA)family is a class of evolutionary conserved polymodal cell sensors responding to external stimuli such as temperature or chemical molecules.In Drosophila melanogaster,the TRPA family consists of four members:DmTRPA 1,Pyrexia,Painless and Waterwitch,which are responsible for the physiological functions of light,temperature,chemical or mechanical sensitivity.Among them,Pyrexia plays an important role in the high temperature sensitivity(>40℃),gravity sensitivity and biological rhythm regulation of Drosophila melanogaster.In addition,Pyrexia,as a Ca2+-dependent cation channel,also plays an important role in regulating the cellular levels of Ca2+.In view of the important function of Pyrexia in Drosophila melanogaster,we carried out both structural and functional analysis of the Pyrexia protein.Firstly,we used the eukaryotic cell expression system to express Pyrexia.By the expression vector screening and the choice of detergents,we obtained well-behaved Pyrexia protein.Then,the cryo-EM samples with good particle distributions were obtained by the quick-freezing technique and optimizations of sample preparation conditions.Finally,we determined the structure of Pyrexia by cryo-electron microscopy single particle analysis with an overall resolution of 3.0 ?.Consistent with other TRP family proteins,the Pyrexia homotetramers are formed through a "domain swap" manner.Pyrexia exhibits a four-fold symmetry around the central ion pathway formed by transmembrane helix 5-6(TM5-TM6)and the intervening pore loop,which is flanked by transmembrane helix 1-4(TM1-TM4)voltage sensor-like domains(VSLD).Apart from this conserved transmembrane core,Pyrexia exhibits several conserved features such as the ankyrin repeat domain(ARD)at the N-terminal and the TRP-like domain at the C-terminal.In addition,a possible Ca2+ density was observed in the ion pathway.We hypothesized that the determined structure of Pyrexia represents an intermediate conformation by comparing with the homologous structures and analyzing the pore size of the channel.The ion selectivity filter of Pyrexia is formed by the electronegative residue Asp691.The upper gate in Pyrexia is formed by residue Glu681,which is wide enough(7.3 ?)to accommodate the partially dehydrated calcium ions.Pyrexia consists of two hydrophobic seals formed by Val720 and Val724,creating an increasingly constricted funnel of which the narrowest point(5.4 A)blocks the conduction of cations.Then,we determined that Pyrexia channels could be activated by Ca2+ and the agonist Iodoacetic acid(IA)using the calcium fluorescence assays.The Ca2+coordination motifs at the bottom of each TM2-TM3 helix facing the cytoplasm and the residue Glu681 are conserved in the TRP family.Mutations of the key amino acids in these regions cause Pyrexia to lose its response to Ca2+.The agonist IA specifically activates Pyrexia channels.Through point mutation,we identified that the conserved residue Cys438 is involved in IA activation.In conclusion,the structural and functional studies of Pyrexia not only facilitate our understanding of the three-dimensional structure of TRP channels,but also enrich our understanding of the channel regulation and ion conduction mechanisms.