Cellular/MolecularSerotoninFacilitatesPeripheralPainSensitivityinaMannerThatDependsontheNonprotonLigandSensingDomainofASIC3ChannelXiangWang, 1,2 Wei-GuangLi, 2 YeYu, 2 XianXiao, 1,2 JinCheng, 2 Wei-ZhengZeng, 1,2 ZhongPeng, 2 MichaelXiZhu, 3andTian-LeXu 21 InstituteofNeuroscienceandStateKeyLaboratoryofNeuroscience,ShanghaiInstitutesforBiologicalSciences,ChineseAcademyofSciences,Shanghai200031,China, 2 DepartmentsofPhysiology,Biochemistry,andMolecularCellBiology,InstituteofMedicalScience,ShanghaiJiaoTongUniversitySchoolofMedicine,Shanghai200025,China,and 3 DepartmentofIntegrativeBiologyandPharmacology,TheUniversityofTexasHealthScienceCenteratHouston,Houston,Texas77030Tissue acidosis and inflammatory mediators play critical roles in inflammatory pain. Extracellular acidosis activates acid-sensing ionchannels(ASICs),whichhaveemergedaskeysensorsforextracellularprotonsinthecentralandperipheralnervoussystemsandplaykeyrolesinpainsensationandtransmission.Additionally,inflammatorymediators,suchasserotonin(5-HT),areknowntoenhancepainsensation. However, functional interactions among protons, inflammatory mediators, and ASICs in pain sensation are poorly under-stood. In the present study, we show that 5-HT, a classical pro-inflammatory mediator, specifically enhances the proton-evoked sus-tained, but not transient, currents mediated by homomeric ASIC3 channels and heteromeric ASIC3/1a and ASIC3/1b channels.Unexpectedly, the effect of 5-HT on ASIC3 channels does not involve activation of 5-HT receptors, but is mediated via a functionalinteractionbetween5-HTandASIC3channels.Wefurthershowthattheeffectof5-HTonASIC3channelsdependsonthenewlyidentifiednonprotonligandsensingdomain.Finally,coapplicationof5-HTandacidsignificantlyincreasedpain-relatedbehaviorsasassayedbythe paw-licking test in mice, which was largely attenuated in ASIC3 knock-out mice, and inhibited by the nonselective ASIC inhibitoramiloride.Together,thesedataidentifyASIC3channelsasanunexpectedmoleculartargetforacuteactionsof5-HTininflammatorypainsensation and reveal an important role of ASIC3 channels in regulating inflammatory pain via coincident detection of extracellularprotonsandinflammatorymediators.IntroductionAcid-sensing ion channels (ASICs) are members of the voltage-insensitive, amiloride-sensitive epithelial sodium channel/de-generin family of cation channels, which have emerged as keysensors for extracellular protons in central and peripheral neu-rons.Todate,functionalcloningstudieshaverevealedfourgenesthat give rise to at least six ASIC isoforms (ASIC1a, ASIC1b,ASIC2a, ASIC2b, ASIC3, and ASIC4) (Lingueglia et al., 1997;Waldmannetal.,1997a,b;Chenetal.,1998;Gru¨nderetal.,2000).In brain neurons, calcium-permeable ASIC1a channels are re-quired for acid-evoked currents and they contribute critically toacidosis-induced neuronal death (Xiong et al., 2008; Sluka et al.,2009; Gru¨nder and Chen, 2010; Wang and Xu, 2011). In periph-eral neurons, ASIC3 channels are important for sensing inflam-matorypain(Mametetal.,2002,2003;Devaletal.,2008;Walderet al., 2011).Tissue injury and inflammation cause acidosis, which is suffi-cient to activate ASIC3 channels and can trigger pain sensation(Steen and Reeh, 1993; Reeh and Steen, 1996; Frey Law et al.,2008). Interestingly, activation of ASIC3 by protons generatesbiphasic inward currents: a large transient current followed by asmall sustained current with little or no desensitization duringcontinued presence of the low extracellular pH (Lingueglia et al.,1997;Waldmannetal.,1997b;Babinskietal.,1999;Bensonetal.,2002). The sustained current is particularly enhanced whenASIC3 is activated by protons in the presence of neuropeptidessuchasFMRF-amideandrelatedproducts(Askwithetal.,2000),a property thought to be essential for sensing prolonged acidosisunder pathological conditions. Previous studies have suggestedthat the sustained currents of ASIC3 channels have significantphysiological or pathophysiological relevance, such as pain per-ception (Dube´ et al., 2005b; Yagi et al., 2006).In addition to acidosis, the inflamed tissues are also enrichedwith other factors, collectively called inflammatory mediators.Together, these factors confer nociception as shown by the con-ReceivedJuly16,2012;revisedDec.19,2012;acceptedJan.12,2013.Authorcontributions:X.W.,Y.Y.,andT.-L.X.designedresearch;X.W.,W.-G.L.,X.X.,J.C.,W.-Z.Z.,andZ.P.per-formedresearch;X.W.,W.-G.L.,andM.X.Z.analyzeddata;X.W.,W.-G.L.,M.X.Z.,andT.-L.X.wrotethepaper.This study was supported by grants from the National Natural Science Foundation of China (Nos. 31230028,91132303, 91213306, and 31170787) and Key National S&T Program “Major New Drug Development” Grant2012ZX09504001-003.WethankDr.M.J.Welsh(HowardHughesMedicalInstitute,UniversityofIowa,IowaCity,IA)forprovidingASIC3andASIC1aknock-outmice.CorrespondenceshouldbeaddressedtoDr.Tian-LeXu,NeuroscienceDivision,DepartmentofBiochemistryandMolecularCellBiology,InstituteofMedicalScience,ShanghaiJiaoTongUniversitySchoolofMedicine,280SouthChongqingRoad,Shanghai200025,China.E-mail:xu-happiness@shsmu.edu.cn.DOI:10.1523/JNEUROSCI.3376-12.2013Copyright©2013theauthors 0270-6474/13/334265-15$15.00/0TheJournalofNeuroscience,March6,2013 • 33(10):4265–4279 • 4265
