血清白蛋白对_7_烟碱型乙酰胆碱受体反应的增强作用_英文_

血清白蛋白对_7_烟碱型乙酰胆碱受体反应的增强作用_英文_ () 文章编号 : 1007 - 6611 2000S0 - 0055 - 05 α血清白蛋白对- 烟碱型乙酰胆碱受体反应的增强作用 7 ()刘青松 ,Co nroy W G , Berg D K 美国加利福尼亚圣地亚哥大学生物学教研室 α(α) 摘要 : - 烟碱型乙酰胆碱受体 受体广泛分布于中枢及外周神经系统 ,参与多种生理功能的调节并与老年性痴呆等疾 7 7 ( ) α病有重要联系 。在培养的鸡胚睫状神经节及大鼠海马神经元上 ,我们发现牛血清白蛋白能大幅度 达,10 倍增强受体电 7 流 ,这一作用不依赖膜电位 ,受体与激动剂亲合力增加也不是直接原因 。再纯化实验及蛋白酶消化实验证明牛血清白蛋白的 作用不大可能是由于与其结合的其他污染分子造成 ,而更可能是由于其本身的作用 。牛 、马 、羊 、狗 、猪等动物的血清白蛋白 有类似的增强作用 ,而鸡 、大鼠 、小鼠 、人的血清白蛋白则无增强作用 ,表明白蛋白的作用有特异性 。不同种动物血清白蛋白 α氨基酸顺序的差异提示我们可能能够用合成的小分子多肽来模拟血清白蛋白对受体的增强作用 ,并以此为线索来发现体 7 内存在的内源性类似物 。 关键词 : 血清白蛋白 ; 受体 ,胆碱能 ; 神经系统 + 中图分类号 : R363 . 2 72 文献标识码 : A αThe enhancement of - conta in ing n icotin ic acetylchol ine receptor 7 response by serum al bumins ( L IU Qing - so ng , Co nroy W G , Berg D K Depa rt ment of B iology , U ni versi t y of Cal i f or ni a S an Diego , L a Jol l a , CA 92093 - )0357 , U S A ( )This paper is dedicated to Professor Q IAO J ian - tian o n his 70t h birt hday Key words : serum albumin ; recep tors ,cholinergic ; nervous system 3 discrete clusters o n t he neuro n surf ace. Alt ho ugh 1 Introduct ion αα Nicotinic acet ylcholine recep to rs co ntaining 2nACh Rs appear to be excluded f ro m po st synap tic77 α() gene p ro duct 2nACh Rsare o ne of t he mo st abun2 densities o n ciliary neuro ns , t hey no net heless are ca72 4 dant neuro nal nACh Rs in t he nervo us system. The pable of generating large synap tic current s. A much αactivatio n of 2nACh Rs generates a f ast desensitizing 7ααless abundant class of recep to rs co ntaining t he , , 3 5 3 ββα(), and so metimes gene p ro duct s 2nACh Rs current w hich can be blocked by snake veno m2bun2 4 2 3 1 αα() garoto xin Bgt . The2nACh Rs have a high cal2 7are co ncent rated partly in po st synap tic densities o n t he 2 cium per meabilit y, w hich has been estimated to be neuro ns , and also co nt ribute to t he synap tic respo nse . 6,20 times mo re per meable to calcium t han to so di2Bot h synap tic stimulatio n i n si t u and ago nist ap2 um. Due to t heir high calcium per meabilit y and di2 plicatio n i n v i t ro no r mally activate o nly a small f rac2 2 ,3 αverse locatio ns, t he2nACh Rs influence multiple 7αtio n of t he 2nACh Rs in C G neuro ns , suggesting 7cellular p rocesses , including enhancement of p resy2 αmany of 2nACh Rs may be silent despite access to 7nap tic t ransmit ter release , regulatio n of co2exp ressed ago nist . Mo reover , E8 C G neuro ns grow n in cell cul2 po st synap tic recep to rs , neurite o ut growt h , and sec2 5 ,6αt ure exp ress substantial numbers of 2nACh Rs 7o nd - messenger cascade . Mo re impo rtantly , recent and yet o ur initial effo rt s to detect Bgt2sensitive , evidence indicates t hat impair ment of t he f unctio n of rapidly desensitizing respo nses f ro m t he neuro ns yield2 α2nACh Rs is clo sely linked to t he pat hogenesis of 7ed very small current s at best , even wit h rapid appli2 several cognitive diso rders such as Alzheimer’s disease catio n of ago nist . These result s imply t hat dynamic and Par kinso n’s disease . regulato ry mechanism co uld exist to resto re o r boo st αThe richest so urces of 2nACh Rs know n to date 7αt he2nACh R f unctio n in respo nse to t he need of t he 76 is t he chick ciliary ganglio n , w hich co ntaining , 10bo dy. In t rying vario us agent s fo r augmentatio n of such recep to rs per neuro n at t he end of develop ment . t he respo nse , we recalled t hat co mmo n bovine serum Recent evidence indicates t hat t he recep to rs are co n2 () albumin B SAhad been repo rted to increase t he re2 3 3 cent rated o n so matic spines t hat are tightly folded into αspo nsiveness of C G2nACh Rs by a mechanism t hat () ato r Burleigh. The rate of solutio n exchange at t he eit her utilizes o r co nverges wit h a cAM P2dependent 7 pat hway. In t he p resent paper , using w hole2cell cell surf ace was estimated by mo nito ring t he liquid reco rding , we have examined t he effect of serum al2 junctio n potential change at an open patch pipet te , αbumins o n t he 2nACh R respo nse f ro m C G neuro ns2 and was fo und to require < 5 ms. Ago nist s were test 7 and rat hippocampal neuro ns in cell cult ure . ed o n cells at 1 min intervals to allow recovery f ro m 2 Material and method desensitizatio n . 2 . 1 Cell p reparatio ns Dissociated C G neuro ns were Membrane current s were amplified and filtered at p repared f ro m embryo nic day 13 chick ciliary ganglia . 1 k Hz using an Axopatch 200 A patch clamp amplifier The ganglia were dissected f ro m t he embryo , and in2 () Axo n Inst rument s Inc. , Fo ster City , CA, digitiz2 ( cubated wit h 1 g/ L collagenase t ype 1 , Wo rt hingto n ( ) ing wit h DigiData 1200B Axo n Inst rument s Inc. , ) (Biochemicalfo r 20,30 min at 37 ?. The dissocia2 and acquired at 2,5 k Hz using Clampex 6 Axo n In2 () ) tio n medium co ntained in mmol/ L : 150 NaCl , 2 . 5 st rument s Inc. o n a perso nal co mp uter . U nless ot h2 KCl , 2 CaCl, 1 MgCl, 10 gluco se , and 10 H EP ES , erwise indicated , data are p resented as x ?s, and ?2 2 x? ( ) p H 7 . 4 wit h NaO H. Following collagenase t reat2 were evaluated fo r significance using eit her t he paired ment , t he ganglia were dispersed to single cells and o r unpaired t2test as app rop riate . ( cult ured in Eagle’s Minimal Essential Medium GIB2 2 . 3 Materials White L egho r n chick embryo s were o btained locally and maintained at 37 ? in a humidi2 ) ( ) COsupplemented wit h 10 % vol/ volheat - inacti2 αfied incubato r . Bgt was p urchased f ro m bioto xins ( ) ( ) vated ho rse serum Gemini and 3 % vol/ vol em2 ( ) St . Clo ud , Flo rida. All ot her reagent s were p ur2 bryo nic eye ext ract . Dissociated cells were used wit hin chased f ro m Sigma unless ot herwise indicated. 6 ho urs of plating and were kep t in a humidified tissue cult ure incubato r wit h 95 % air/ 5 % COat 37 ? un2 2 3 Results and discussion til use . μ20 mol/ L nicotine to C G Rapid applicatio n of Electrophysiology Whole2cell patch2clamp recordings 2. 2 αneuro ns activates bot h 2nACh Rs , w hich generate 73 3 were obtained from isolated ciliary neuro ns as p revio usly αα f ast2desensitizing , Bgt2blockable current ; and 21 described. Patch pipet tes were p ulled f ro m t hin2 α nACh Rs , w hich generate slow2desensitizing ,Bgt2re21 () ( walled 1 . 5 mm O . D . bo ro silicate glass t ype N51 , sistant current . Incubatio n of bovine serum albumin ) Drummo nd Scientific , Broo mall , PAusing a Sut ter () B SA to f reshly dissociated E13 C G neuro ns p ro2 αduces a large increase in t he w hole2cell 2nACh R re2 Inst rument s P287 pipet te p uller , and had resistances 7μ( ) spo nses to 20 mol/ L nicotine Fig 1. In f act , t he Ω of 1, 1 . 5 Mfo r perfo rated patch pipet tes and 2, current s are so large under t hese co nditio ns , t hey usu2 Ω 2 . 5 Mfo r co nventio nal patch pipet tes. The int racel2ally sat urate t he amplifier ; to avoid t his we of ten ad2 ( lular solutio n , unless stated ot herwise , co ntained in just t he clamp potential to - 20 mV instead of t he ) mmol/ L : 140 CsCl , 2 MgCl, 2 E GTA and 10 2 usual - 60 mV to measure t hem. However , no sig2 ( ) H EP ES , p H 7 . 2 wit h CsO H. Series resistance fo r bot h reco rding co nfiguratio ns was in t he range of 5, nificant difference was detected in t he mean peak am2 Ω 10 M; series resistance co mpensatio n of 80 % was applied. The exter nal solutio n fo r w hole2cell reco rdings () was in mmol/ L : 150 NaCl , 2 . 5 KCl , 2 CaCl, 1 2 ( MgCl, 10 gluco se , and 10 H EP ES , p H 7 . 4 wit h 2 ) NaO H. Nicotine and ACh were applied via a rapid ( applicatio n system as p revio usly repo rted Zhang et ) al . 1994. Briefly , co nt rol and ago nist2co ntaining so2 lutio ns were delivered o nto t he cells f ro m a linear ar2 (ray of f used glass t ubes 0 . 35 mm I. D . and 0 . 45 mm ) O . D . ; Polymicro Technologies , Phoenix , A Z . Flow of solutio n t hro ugh t he individual t ubes was in2 Fig 1 Whole2cell patch2clamp recordings showing potentiatio n αμof E13 C G2nAChR respo nse to 20mol/ L nicotine in t he ab2 7duced by gravit y feed and regulated by solenoid valves ( ) general valve , Fairfield , NJ . Movement of t he t ube μ( )( ) sence lef t t races or p resence right t racesof 100 mol/ L () () array was mediated by a piezoelect ric bender act uato r BSA. A. Clamped at - 20 mV ; no Bgt . B. Clamped at α - 60 mV ; 100 nmol/ LBgt p resent .( ) P2803 . 40 , Polytec P Ico nnected to a voltage gener2 3 3 α( Additio nal experiment s indicate t he potentiati ng plit ude of t he 2nACh R w hich is not sensitive toα2nACh R respo nse was mo re effect of B SA o n t he 7) αBgtrespo nse in t he p resence and absence of B SA likely a direct o ne . It is not mediated eit her by calci2 ( ν) () [ 42 ?5s 44 ?6pA/ p F ; n = 12 cells each , su g2 um influx o r by G p rotein2linked recep to rs since it is gesting selectivit y of B SA actio n . B SA p ro duces a not affected by inter nally dialyzing t he cell wit h BA P2 αdo se2dependent potentiatio n of 2nACh R respo nse , 7 μhalf2maximal stimulatio n is achieved at , 10 mol/ L βγTA , GD P22S , o r GTP22s. These result s , toget her ) (Fig 2 . The B SA effect is rapid in o nset and re2 wit h a rapid o n and off time co urse , indicate t hat t he ( ) versibilit y Fig 3. The effect of B SA is voltage2inde2 effect is unlikely to employ mechanisms utilized by pendent , B SA yields similar potentiatio n at negative PACA P w hich has recently been show n to affect re2 and po sitive holding member potentials. The rectifi2 8 cep to r f unctio n , t ho ugh much mo re mo destly . catio n of t he w hole2cell nicotinic respo nses at po sitive αMo reover , we find t hat t he B SA effect extends to2 7potential is not altered. The increase of t he affinity of nACh Rs exp ressed by rat hippocampal neuro ns and to ago nist wit h t he recep to r is not impo rtant fo r t he B SA αchimeric 2nACh R/ 5 - H Trecep to rs exp ressed by 73 enhancement , since a much less extent of potentiatio n t ransfected cells. This lat ter result raises t he po ssibili2 ( μ) were seen w hen low co ncent ratio ns < 5mol/ L of t y t hat t he B SA effect is exerted by a direct interac2 nicotine were used. tio n wit h t he ext racellular amino2ter minal po rtio n of t he recep to r . Albumins are noto rio usly know n to bind a num2 ber of substances , w hich may be co2p urified during p urificatio n. The enhancement of B SA co uld be achieved by being able to deliver an enhancing f acto r o r abso rb an inhibito ry o ne . Recently we came acro ss a repo rt t hat t he ant helmint iver mectin might have a Fig 2 Co ncent ratio n2dependence of BSA stimulatory effect o n 9 similar effect and t hat it can bind to albumins . t he peak nicotinic respo nse of f reshly dissociated E13 C G neu2 Co nt rary to p ublished st udies wit h heterologo usly ex2 ro ns. Current s are normalized for t hose obtained in t he absence 9 of BSA. p ressed recep to rs, however , we find t hat applica2 μtio n of iver mectin eit her at 10 o r 30mol/ L p ro duces αo nly a slight enhancement of native 2nACh R re2 7 spo nses in C G and , mo re impo rtantly , t he enhanci ng effect of B SA does not arise f ro m bo und iver mectin2 like substances. Aceto nit rile ext ractio n of B SA , w hich removes such substances , does not reduce t he B SA effect . No r does t he aceto nit rile ext ract , w hich sho uld be enriched in such co mpo nent s , display an enhancing effect . Thus abso rp tio n of iver mectin2like substances by certain albumins is not likely to explain t he selective enhancement we o bserve . It is also unlikely t hat B SA achieves it s enhanc2 Fig 3 Rapid o nset and reversal of BSA potentiating effect o n ing effect by abso rbing inhibito ry co mpo unds. Co m2 α2nAChR nicotinic respo nses of ciliary ganglio n neuro ns. C G 7po unds t hat inhibit neuro nal nACh Rs do exist , e . g. μneuro ns were challenged wit h rapidly applicatio n of 20 mol/ L μnicotine af ter t he indicated exposure to 10 mol/ L BSA. The 11 10 E2arachido nic acidand p ro staglandin but t hese time of exposure to BSA was caref ully co nt rolled by delivering it are unlikely to explain t he effect because o nly certain f ro m app rop riate barrels of t he rapid applicator . Wit hin 20 s , αalbumins are capable of enhancing t he 2nACh R re2 7t he f ull potentiating effect is realized ; it can be washed out wit hin a few seco nds. spo nse t ho ugh all can scavenge f at t y acids and related ( ) molecules see next paragrap h . Mo reover , t he en2 hancement is rapidly reversible even fo r f reshly disso2 ?So me , but not all , albumins p ro duce t he latio n , ciated and isolated cells ; t his wo uld mean t hat t he enhancement , suggesting a specific molecular interac2 3 3 cells wo uld co ntinually have to generate t he inhibito ry ααtio n , ?2nACh Rs , but not 2nACh Rs respo nd , 7 in such abundance as to re2establish inhibitio n wit hin indicating specificit y in t he target , and ?Endo geno us seco nds of removing t he B SA despite t he co ntinuo us co mpo nent s such as lynx1 can have mo dulato ry effect s 12 rapid perf usio n p rovided by large bo re applicato r o n neuro nal nACh Rs by way of p recedent t ho ugh pipet te bat hing t he cell . t he effect is not nearly as ro bust as t hat seen here . We have fo und t hat albumins f ro m bovi ne , The alter native po ssibilit y is t hat albumin effect is ( sheep , dog , pig , cat , and almo st as effec2 ho rse , serendipito us and involves a site/ mechanism fo r )rabbit p ro duced equivalent 62fold stimulatio n tively w hich no endogeno us activato rs exist . The o utco me μof t he respo nse w hen applied at 10 mol/ L . In co n2 wo uld also be interesting and usef ul : if t he effect is t rast , ovalbumin and human , chicken , rat and mo use exerted directly o n t he recep to r , it co uld p rovide new albumin had no effect . Albumins f ro m different αst ruct ure2f unctio n info r matio n abo ut 2nACh Rs. 7species display co nsiderable sequence variatio n . It is Even mo re interesting wo uld be t he po ssibilit y t hat very likely t hat a specific amino acid sequence in B SA t he albumin effect was mediated indirectly. In t his αis respo nsible fo r t he enhancement of 2nACh R re2 case , t he f act t hat a signaling system existed capable 7 spo nses. Co mparing active and inactive albumins will αof p ro ducing a 102fold increase in t he 2nACh R re2 7reveal info r matio n of candidate sequence respo nsible ( )spo nses regardless of t he no r mal mo de of activatio n fo r t he potentiating effect . wo uld p rovide eno r mo us incentive to understand t he We have extended o ur p roteolysis analysis by us2 mechanism and deter mine t he kinds of p hysiological ing p ublished p rocedures to achieve limited B SA di2 sit uatio ns t hat are made use of i n v i v o. gestio n wit h pep sin under co nt rolled co nditio ns. We A ck now led ge m en t : L I U Q i n g - son g w is hes to find t hat t he carbo xyl half of B SA co ntains t he activi2 t ha nk Prof . Q IA O J i a n - t i a n f or his con t i n uous t y and t hat f urt her digestio n to a series of small pep2 g ui d a nce , encou ra ge m en t a n d hel p . tides still allow s t he activit y to be recovered. Full di2 Ref erences : gestio n kills it . Interestingly , under t hese p roteolysis 1 ] Zhang ZW , Vijayaraghavan S , Berg D K. Neuro nal acet ylcholine co nditio ns , t he carbo xyl tail is p reserved. This is a recepto rs t hat bind alp ha - bungaroto xin wit h high affinit y f unc2 tio n as ligand - gated io n channelsJ . Neuro n , 1994 , 12 : 167, st ro ng co ntender fo r t he active co mpo nent because it 177 . is expo sed o n t he intact B SA and co ntains several Bert rand D , Galzi J L , Devillers - Thiery A , et al . Mutatio ns at 2 ] amino acids unique to t he“co mpetent ”albumins. The t wo distinct sites wit hin t he channel do main M2 alter calcium per2 active pep tide will be isolated by HPL C , tested , and αmeabilit y of neuro nalnicotinic recepto r J . Proc Natl Acad Sci 7 synt hesized fo r co nfir matio n of activity. U SA ,1993 , 90 : 6971,6975 . In summary , we have fo und t hat certain albu2 Shoop RD , Marto ne M E , Yamada N , et al . Neuro nal acet yl2 3 ] mins can rapidly and dramatically increase t he w hole2 αsubunit s are co ncent rated o n so matic choline recepto rs wit h 7 αcell nicotinic 2nACh R respo nse . There are t wo 7spines fo r synaptic signaling in embryo nic chick ciliary ganglia J . J Neuro sci 1999 , 19 : 692,704 . classes of explanatio ns fo r t his result , and bot h merit Zhang ZW , Coggan J S , Berg D K. Synaptic current s generated by vigo ro us investigatio n . Mo st interesting is t he po ssi2 4 ] αneuro nal acet ylcholine recepto rs sensitive to- bungaroto xin J . bilit y t hat t he co mpo nent albumin p ro duces t he effect Neuro n ,1996 , 17 : 1231,1240 . αby mimicking an endogeno us mo dulato r of 2 7Ravdin PM , Nit kin RM ,Berg D K. Internalizatio n of alp ha - bun2 αnACh Rs. 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