3P-199 Isolation of temperature sensitive mutants of the mechanosensitive channel MscL in Escherichia coli(The 46th Annual Meeting of the Biophysical Society of Japan)
3P-199 大腸菌機械受容チャネルMscLの温度感受性変異体の単離(生体膜/人工膜・興奮,チャネル,第46回日本生物物理学会年会)

Kenjiro Yoshimura, Noriko Kasai
2008 Seibutsu Butsuri  
TheBiophysicalSociety of Japan General IncorporatedAssociation Shllrmdza synthess sygtem (TrangdirLLt) prepared iTorn SMI cclls usTng erthLT canmL pancreatic m-rosomL or insect cellts microsome lysate ffith ¢ r lysate expreggLd BK chdnntlg effZcicntly Wle a]so rLport the cfTLLts of DTT on BK LhdnneL actrvity and its mcorporatien into the LLpid bdayeT This cell tree system denved from insLct cellg i$ potcntidl]y a ugLfu1 tool to gynthcsiie eukdryete 'refen:v nydkd N}oto Jlwan It ig pt eU knewn
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It ig pt eU knewn that argirune vasotoun {iWT) stimuJates thL godium reabsorption by mcieasmg the nuniber ot lrNaC rn the aptcal memhrane of renal epithelJal eells Tt is also ind!cated that Cl regulates Lell functiolls suLh as cell growth and neumte elongaaon HoweseT, the efibLt of Cl o]i the AAJTstir["ilated I]NaC mLdiatLd current is unclear In the prLsent study we exammed efiect ot Ll on aLtEvity of AVT stimutated ENaC m rtnal epithelial A6 cells AXrT signiiicantly inLreased the ]NdC medLated Lurrents m A6 LLI]s Lowenng the Cl conLcntrition efthc dpical soluuon by replacmg Cl with gluconate d!rninished the stimulatory action ef AVT on the ENaC mediated current while the dimmishmg effect ot lowermg Cl on the t"VT aLtaon vvag not obberved undLT a low Ci Londmon tor the basolatcral solutien We theiefore exarnmed effect oflowermg the apicat Cl concentration on acavity (Ml'o) of ENaC by cell attaehed patch clamp techmque Undei a low apiLd] Cl condition, the cunLnt Noltage relationship of ENdC Khfied ILftward about 50 mV and IW]o at ENaC "as lower than that undei a notinal condition In addition thesL effeets ol lowmg Ll on ENaC were also observed under a low Ll pipLtte hoJution rvith a normn1 bath setution ThcsL resutts guggLst that thL apiLal chlondL ion is a key molLeale for iWT to sho" itg stimn!atory action on the ENaC mediatLd sodium rLabsorpzion m rLnal epithehal Letls Supported by jtsPS l9S90212 20390060 The Salt Science R"edrch Foundation 0736 Og37 and KI'UM RAS prqecr We mvestigated the dynamics ofK iong thTough an amion dopcd caibon nanotube (ANT) in terms ot thL molecu]ar dyndrmcs (MD) sm]ulation The free enugy Lnergy and Lnti opy profiILg abf)ut thL ion pci meainon through ANT aTe caLculatLd lt is foumd that the free energy prufite is mostly determincd by the cntropy term that is the entaopv dormnates the dynamics of ion permeation through ANT The molecular ongm of the entiopy is revLaledi the cangtrdtnt of thL oriLntdt]on ot a water moleculc makes the entropy barricr at thc entranee of ANT rhe depLndency of thL free energy enLrg), atid entiopy oll the charge on ANT is dlso analyzed ThL Tate of ion pe"nLation is tound to sengitivLly depend on {hL charge o" ANT The rmtrrrum channel conductancc is obtainLd at a certain charge )alue where the bdlance of the e]ergy and entrepy makes the lorv free eierg} barrier We also gtudy the ion pL"nLition mecharusm through the K chaimd Kvl 2 embLdded in the DOPC membra:]L The role ofthe coneentration Js Jnvestigdted ind the MD sirnulation with the electnc held to obgcrvu the ion tluxes is pertormed Ihe gLieral rnechanisrns ofion permeation foumd in the modLa gystem aTe txamined for rhis rLaL system 3P-201 .reentlttsigMtl)(RVD)[=elk7TelereZ"tt6anmepmCl ,XE{ET i:pmfiapa)-tiV-JVLLTeefi2t6 PhygiologiLal imporLaiiLL efregulatory volume dLLrLase reduLoon in mtracelluldr C! concentratron acting as an mtraeellular signaling Hiroaki Wvatain laumi MJsato and }b"hmon WbFunaAa OepartmLnt of Vihlecutar CLii Pig,viology Graduate Schooi of Ldbdicai Slrienee eveto PFdecturai Onlvensle, oj MbdtLalnoj 3P-198 F7)iX7t£ tr)i7).ffAfi).rkeMLSftlagV7/;;)ieegesCD esfireeEfieut Stmcture imction analysis of type 1 ryanodJne recepzoi by transpeson based T tndom lmsertion Tkekdtshi Witropama (ij lhetop aKbbaea,hi a) iltiw lldsh"ama a) Nagemt Mirebayasht rly thshryuki Kitai (Z) and Tlrdavkt 11ikcthagJ C2) (1 Dept Pharmacotogy thintcndb -lty Scheel ofAdLdiLme 2 H)rosima LiniL Graduatc Schoei ofAdmnced grienees ofMinneij Tb,pe 1 Ryanedine receptor (RyR1) ib an mtraLLIIuldr ( ai releage Lha"nel m tlx barcoplasmic Teticulum in skeletal nmscle and plays an importaiit ]ole in exutauon contruLtlon coupl]ng A relativeiy small C termlnal rLglon (500 1,eOO rLsidues) of the RyR constitutcs dn ion channel and the other N tLrmi"aE region fo"ms a1argL cytoplagmiL agsembly {4 OOO450U residueg) whieh serves as the regulatorydomam The gtructure function ielatlontshlpofRyRl however remams u"clear especiaJTy !n the Lytop!asrnie assLTnbly due to its laTtse sizL A tanspogon basLd random mseruon approach is a powerfu1 toel to study the structure functlon rclainonshlp of 1argc pTotems I" thig study we apphed this method to RyRl by msertmg GtP usmg Tn5 tra]sposon Tbe cDNA for GrP tagged RyRl wdb transteckd in HEK293 Lel]s and the expiLbgion was confirmed by greLn fiuorescencL and thc a-ta RyRl dntibody Ca2 reiease Lhannel funcinon was exammed by eaffeme illduced Ca? idLdse riH]ryanodinL bmdmB and ginglL Lhannel recordmgs The GFP tagged RyRl was affi]ity puTitied by FKBP12 and thL structure vifas aLtermmed by negative gtaming election microseopy We are curtently trying to idLntify the Lecation uf GFP in the threL d]menso]al gtrueturL of RyRl by nanogold labekng The strueturL funct]on rclatiollslHps ofRyRl will be drscussed 3P-199 XmaeetstuftnyTt,*JbMscLoAkptfttsgRosOeees lsolation of tempei aturL sLnyuve mutanLs o{ the mechanogensitive ehannel MseL mEsLhenchiaeoit Kbniirv lhsh]muFa (t) andthnko KLthat Cl) CV DtvStr:tLtBzosa 2 Dp Biol &i CTtaelate Schoot ofl de and Environntentag S"Lnces bniv ofnukub4) Regu]atory volume decrease (RVD) occuTs after hypotomctty caused cell swellmg RYD ls caubLd by actlvdtiom ot lon Lhdnnels and trdnsportLrs which caubL LfTluxes ol K', Cl and II20 IL ulmg to cLll shTnkagL ReLently wL sho"ed Lhat hypetonicity smmulated transeprthelzal Na reabsorp"on via elevatien ot epithelial Na channd LxprLgsioi in iLna1 epitheha A6 Lells m an RVD depeiidLnt imanner and that iLduLtion ot intiacLilular Cl collcentration C[Ct1) stimulated the Na' reabsorption ThesL suggest that RVI) vveuld rLvLahts stimuldtory act.on om the Na' Teabsoq]noll by reducmg [C]1 Howei er dieTeduLtion of [Cll durmg RVI) hds not beLn dLfimtely anaJyzed due to techmcal dificultieg mioked m hahde sensLtiie fiuorebcemt dyes Tn the present studM we have deieleped a new method tor the mcaguTernent of [Clr change du"ng RVI) by usmg d fiou Lytometer with a haltde specitic fluorescem dyL MQAE ThL [C!1, in A6 Lells in an ssetoniL medium way 43 6 -3 1 mM The R]VD taused dragoL diminution of [Cl 1 (fiorn 43 6 to 1O g mM) Under RVD blocked conditions with NI'PB {Cl chdmiel blocker) or qumi"e CK' Lhannel bloLkcT) we did not detect the iLduLtion of [C L1 Based en these observations, -e cenc]ude that one of the physiologiLal signifieances of RVD is the reduLtion ot [Cll, and that R]JD shows its acinen NLa redue"on or[Cl 1 actmg as dn intracLIIular signal regula"ng LeLlulaT phybioLegtLa! funLuons SuppeTted by JS?S 195g0212 19790168 20390060 and Salz Science Reseaich Foundation 0736 Og37 MgcL the meLhanosentsit-,e channLl of iaige condactdnee ot E [ok is a homopentamier of a subumt thdt has twe trdnsmLmbrane htlicLs Ml and M2 MscL is gatcd drrectly by rnembrane tension but 1ittle is tcno"m about thc btmctural bagcs ot the encrgetic barmer on gatJng In this stLidy the rLgion iesponsible toi the ga"ng bamer was surveyed by Tsolatmg tLmperature sensitiLL (TS) mutants Mutant MscL was generated by random mutdgLnLus and scr"ncd for the mutdnts that depressed the eell growth at 42 OC but not at 25 OC in compamson witb wild type MsLL wr so far have obtamed 25 TS mutdnts out ot approximately 43 OOO transformants Although thL tempeiaturt sLns]tivity vaned a:nong nmtants the cell expresyng G22S, 125M and [92T MsLL showed severLgt ternperatuu e sensrtivity amd hardly grew at 420C GIy 22 and Ile 25 arL ioLated m Ml whereas Ile 92 is loeated in M2 These thiLL rLsidues are loLdtLd closely mside the MscL crystal stmcture and do met faLe the membranu Iipid The presLllt resQlt indiLdtes that thL Lhange in thL intLraction among these three residues dcLTLatsethe ga"ng bdiTier of MscL 3P-202 O7:-F+tXdipmwautneiateasfk*JVMSCI tMSC3Ovees Chatacteriza"on of the intracel!ular mechanesepsit-e channel MSCI and MSC3 mC7itantydbmonas lhshitaka Nlakavaman) KLnta Fayue 5okabe MLTyahmo(]34) KLnJtro lhshimu"allny a 5tTwe BtesLten"" Graduate SLhoot of Lilb Envxron Sbtence Liniv oj nuAuba 2fCORPtsORST CeU 1fechanorenrlng JST 3Dept Plyviol Nagopa (imv Graduate Schoel of Mledicai 4 paattonat instiittteforPlysmtogical SLxencey MLchamosensitivL channel ig ]mportant for the cell to sLnge to a variety of mechanicaE stmiuli suLh as osmotiL piessure touLh and gravity The bacteTidl mechdnobensit]ye channcl ofsmall conduLtance MgLS se-es as sdfety valve to protect the cell frem h}po osmotic shock GenLs that encediL MscS hke piotem also tre present m some eukaryotLs Chiantydibmonay possu"es thiee geneg encodmg MsLS 1ikL protLin (MSCI MgC2 MSC3) "ie hase recLntly reported thai MSCI is a mechanosengiLive channel present m the mtracellular spacL of C.hlampdomonas SmcL thL locahzatmn Icmeticg ana thc ion gekLtivity ofMgCl arL diffeient trom MseS 1ike proteins it ts possible that the MscS homologues of Chlamydomonas have the imction d"ersity trom ) Lolt Mseg To explore the functional difiLrLnLe arnong maltiple MscS homologues -e charactenzed MSC3 and compared the pToperties wrth MgC T MSC3 "ag expressed heterogeneously m C{vk cells and the chamel activity was analyied by patch clamp SVheii a nLga"ve pressurc was app!ied to tl]e patch membrane the cbmmnel current wrth a conduetaice of O 3 ng was elicited As 1ilce MhCl MgC3 displayLd pronounced hysteresis MSC.3 opened at high prLssure when thL pressurL wds ]ncreased whLreas MSC3 closed at iow pressure virhen the pressuiL vLas decieasLd Antibodieg agamst thL L trboxyl termmus ofMgC3 rLLognized small spDts in the cytoplasm and the chloroplast These findings suggest that both MSC1 tnd MSC3 is mLchanosensitive chapnel m the inti ace] tular spdLe of Chlaop clomonas 3P-200 EfJVfftt?tJ>tK'f-1#kJVI:es[t6tftviieeeesO-r.tsff S158 NII-Electronic
doi:10.2142/biophys.48.s158_3 fatcat:vlzmjpr4g5dfzmqyrplcn34xyq