LETTERS. Nicotine binding to brain receptors requires a strong cation p interaction

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1 Vol Mrch 2009 doi: /nture07768 icotine inding to rin receptors requires strong ction p interction Xinn Xiu 1 *, yss L. Puskr 1 *, Ji A. P. Shnt 1, enry A. Lester 2 & Dennis A. Dougherty 1 icotine ddiction egins with high-ffinity inding of nicotine to cetylcholine (ACh) receptors in the rin. The end result is over 4,000,000 smoking-relted deths nnully worldwide nd the lrgest source of preventle mortlity in developed countries. Stress reduction, plesure, improved cognition nd other centrl nervous system effects re strongly ssocited with smoking. owever, if nicotine ctivted ACh receptors found in muscle s potently s it does rin ACh receptors, smoking would cuse intolerle nd perhps ftl muscle contrctions. Despite extensive phrmcologicl, functionl nd structurl studies of ACh receptors, the sis for the differentil ction of nicotine on rin compred with muscle ACh receptors hs not een determined. ere we show tht t the 42 rin receptors thought to underlie nicotine ddiction, the high ffinity for nicotine is the result of strong ction p interction to specific romtic mino cid of the receptor, TrpB. In contrst, the low ffinity for nicotine t the muscletype ACh receptor is lrgely due to the fct tht this key interction is sent, even though the immedite inding site residues, including the key mino cid TrpB, re identicl in the rin nd muscle receptors. At the sme time hydrogen ond from nicotine to the ckone cronyl of TrpB is enhnced in the neuronl receptor reltive to the muscle type. A point muttion ner TrpB tht differentites 42 nd muscle-type receptors seems to influence the shpe of the inding site, llowing nicotine to interct more strongly with TrpB in the neuronl receptor. ACh receptors re estlished therpeutic trgets for Alzheimer s disese, schizophreni, Prkinson s disese, smoking cesstion, pin, ttention-deficit hyperctivity disorder, epilepsy, utism nd depression 1. Along with solving chemicl mystery in nicotine ddiction, our results provide guidnce for efforts to develop drugs tht trget specific types of nicotinic receptors. icotinic cetylcholine receptors (nachrs) comprise fmily of $20 homologous sutypes tht medite fst synptic trnsmission throughout the centrl nd peripherl nervous systems 2. The neuronl receptors re found in the centrl nervous system (CS) nd utonomic gngli. f these, the sutype most strongly ssocited with nicotine ddiction nd the trget of recently developed smoking cesstion drugs is termed 42 (refs 3 7). The high nicotine ffinity of 42 receptors, when comined with the ility of nicotine to cross the lood rin rrier nd its fvourle phrmcokinetics, llows nicotine t the sumicromolr concentrtions in tocco smoke to ctivte cutely these receptors, providing rewrd, cognitive sensitiztion nd perhps other effects. In ddition, the highffinity interction llows smoked nicotine to ct s n intrcellulr phrmcologicl chperone of 42 receptors, leding to the upregultion of receptors thought to underlie effects of chronic exposure 6,8. In previous studies of the nachr of the neuromusculr junction (muscle type), we showed tht n importnt contriutor to ACh inding is ction p interction to specific tryptophn (clled TrpB, residue 149, Fig. 1) 9. These results were susequently supported y the importnt series of crystl structures of ACh inding proteins (AChBP) 10,11. These structures reveled the romtic ox structurl motif of Fig. 1, nd the ligning residues re predominntly romtic throughout the Cys-loop fmily of neurotrnsmitter-gted ion chnnels. In other Cys-loop receptors, ction p interction etween the nturl gonist nd one of the romtics is lwys seen, lthough its precise loction vries 12. Interestingly, when nicotine ctivtes the muscle-type nachr, there is no ction p interction 13, consistent with its reltively low ffinity for this receptor. This suggested tht ction p interction could discriminte etween highffinity neuronl receptors nd low-ffinity muscle-type receptors. owever, sutle effects must e involved, s the nachrs of the CS nd neuromusculr junction re homologous throughout most regions of sequence nd re essentilly identicl in the immedite vicinity of the gonist inding site (Supplementry Fig. 1). 153 Loop A Loop B Loop C Loop D TrpB Figure 1 The inding site of AChBP, thought to resemle tht of nachrs. Shown re the four principl loops tht define the inding site 2. Also highlighted re TrpB (149) studied here; its ckone cronyl (green str); nd the cron on position 153, which hs lso een mutted here. ote tht loop C contriutes two romtic residues; the other loops ech contriute one. The imge is of Protein Dt Bnk file 1I9B (ref. 10). 1 Divisions of Chemistry nd Chemicl Engineering nd 2 Biology, Cliforni Institute of Technology, 1200 Est Cliforni Boulevrd, Psden, Cliforni 91125, USA. *These uthors contriuted eqully to this work. 534

2 ATURE Vol Mrch 2009 ere we descrie studies of the 42 neuronl receptor. We find remrkle ltertion of inding ehviour: oth ACh nd nicotine mke strong ction p interction to TrpB. In ddition, hydrogen ond from nicotine to the ckone cronyl of TrpB tht is wek in the muscle-type is much stronger in the 42 receptor. Tken together, these two noncovlent interctions fully rtionlize the differentil ffinity of nicotine in the rin versus the neuromusculr junction. A ction p interction etween drug nd receptor cn e reveled y incorportion of series of fluorinted mino cid nlogues (Fig. 2); consistent trend in receptor response indictes inding interction. Such n experiment is enled y the nonsense suppression methodology for incorportion of unnturl mino cids into receptors nd chnnels expressed in Xenopus oocytes. Although we hve found the nonsense suppression methodology to e rodly pplicle 14,15, implementing the methodology for study of the 42 neuronl nachrs proved to e especilly chllenging, requiring new strtegies. The 42 receptors re expressed in Xenopus oocytes t indequtely low levels for nonsense suppression experiments. owever, recent studies showed tht the Leu99Al (L99A) muttion in the M2 trnsmemrne helix of the 4 suunit gretly improves expression without ltering the phrmcologicl selectivity of the receptor 16. (In Cys-loop receptors, the highly homologous M2 sequences re often compred y numering from the cytoplsmic end, termed position 19.) Therefore, ll studies of 42 descried here included this muttion. As with other muttions of L99, the L99A muttion lowers the gonist concentrtion for hlfmximum response (EC 50 ) y influencing receptor gting in wys tht re firly well understood nd tht do not distort the present nlysis of the inding site (some 60 Å from the 99 position) 17,18.In ddition, previous studies of the muscle-type receptor used comprle muttion t L99, nd control experiments estlished tht it did not lter inding trends 9,19. The nachrs re pentmeric. The muscle-type receptor hs precise stoichiometry of (1) 2 1cd. owever, the 42 receptor cn hve vrile stoichiometry. In prticulr, there re two forms of 42, (4) 2 (2) 3 nd (4) 3 (2) 2, which we refer to herefter s A2B3 nd A3B2, respectively 8,20,21. Agonist inding sites re t the pproprite interfces. The A2B3 form hs higher sensitivity for nicotine nd my e upregulted during chronic exposure to nicotine; our studies hve focused on it. Controlling the rtios of messenger RAs injected into the oocyte cn relily control suunit c c C 3 C 3 + C 3 + C 3 ACh icotine d ,,c,d = = F,d = F,c,d = F,,c,d = F = C = Br Trp F-Trp F 2 -Trp F 3 -Trp F 4 -Trp C-Trp Br-Trp + Figure 2 Agonists nd unnturl mino cids considered here., Structures of ACh nd nicotine., Unnturl mino cids considered here. If not indicted, n,, c, or d group is. Br, romo group; C, cyno group. c, The ckone ester strtegy for modulting hydrogen ond. stoichiometry in the wild-type receptor. owever, in nonsense suppression experiment, the suunit tht contins the stop codon where the unnturl mino cid hs een incorported cn show low nd vrile expression levels. Therefore we sought second, independent indictor of the stoichiometry of the 42 receptor. We now report tht the A2B3 nd A3B2 forms of the 4(L99A)2 receptor show mrkedly different rectifiction ehviours. As indicted y either voltge rmp or voltge jump experiments, A2B3 is sustntilly more inwrd rectifying thn A3B2 (Supplementry Fig. 2). Thus, in ll our experiments with unnturl mino cids, the stoichiometries of mutnt receptors re monitored y mesuring current voltge reltions with voltge jumps. For ech mutnt receptor studied, we determined the frction (outwrd current t 170 mv/ inwrd current t 110 mv), nd vlue #0.1 estlishes the desired A2B3 stoichiometry (Supplementry Tle 1 nd Supplementry Discussion). With these methodologicl developments in hnd, incorportion of unnturl mino cids into the 42 receptor ecomes fesile (Fig. 3). As shown in Supplementry Tle 1 nd Fig. 4, compelling fluorintion trend is seen for oth ACh nd nicotine t TrpB of the 42 receptor. This is in contrst to the results t the muscletype receptor, in which no such trend is seen for nicotine ctivtion. Further support for n importnt ction p interction for oth gonists is provided y the lrge perturtion induced y cyno (C) group which is strongly dectivting in ction p interction compred to romo (Br) group, which is roughly isosteric to cyno group ut much less dectivting. We hve lso evluted other residues tht constitute the romtic ox of the ACh inding site (Supplementry Tle 1). The results for 42 very much prllel our previous findings for the muscle-type receptor (Supplementry Discussion). This indictes tht it is specificlly the interction with TrpB tht discrimintes the two receptor sutypes. The EC 50 vlues reported here represent mesure of receptor function; shifts in EC 50 cn result from chnges in lignd inding nd/or receptor gting properties. By scriing the results to ttenution of ction p interction, we re effectively concluding tht it is lignd inding tht is eing modulted y fluorintion, ut tht conclusion is not incontrovertile. To resolve this miguity, we evluted the gting ehviours of key receptors using single-chnnel recording. For the wild type nd the receptor with 5,6,7-trifluorotryptophn (F 3 -Trp) t TrpB, we compred the proilities tht the chnnel is open (P open ) t nicotine concentrtions tht evoke hlfmximl mcroscopic stedy-stte currents (EC mm nd 1.2 mm, respectively). Any differences etween the two P open vlues must result from differences in gting ehviours. As suggested y Fig. 5 nd s confirmed y further single-chnnel nlysis (Supplementry Fig. 3 nd Supplementry Discussion), the wild-type nd mutnt receptors hve P open vlues tht re essentilly indistinguishle. Thus, the shift in EC 50 for F 3 -Trp is primrily, if not exclusively, consequence of chnges in inding. Fluorintion of TrpB of the 42 (A2B3) receptor primrily hs n impct on the sensitivity to nicotine y decresing nicotine s ction p interction with this residue. These results indicte tht nicotine is positioned more closely to TrpB in the 42 gonist inding site thn in the muscle type. This suggested tht nother nicotine-inding interction could lso e ltered. An importnt chemicl distinction etween ACh nd nicotine is tht only the ltter cn ct s hydrogen ond donor, through the pyrrolidine 1 - (Fig. 2). Exmintion of the AChBP crystl structures (Fig. 1) 22 suggested tht the ckone cronyl ssocited with TrpB could ct s the hydrogen ond cceptor, nd severl groups hve shown the importnce of this interction Previously, we proed this potentil hydrogen ond in the muscletype receptor y replcing the (i 1 1) residue with its -hydroxy nlogue (Fig. 2c). This converts the ckone mide to ckone ester, which is well estlished to e sustntilly poorer hydrogen ond cceptor. In the muscle-type receptor, this chnge rised the 535

3 ATURE Vol Mrch I ormlized µa 10 s Figure 3 onsense suppression in the 42 receptor. Shown is wildtype recovery experiment, in which Trp is incorported t the TrpB position., Representtive trces of voltge-clmp currents. Brs represent nicotine EC 50 y modest fctor of 1.6 (ref. 25). We now find tht for precisely the sme chnge in the 42 receptor, the nicotine EC 50 increses 19-fold, reltively lrge effect for such sutle muttion Recll tht the ckone ester sustitution does not destroy the hydrogen ond, it simply ttenutes it. otly, ACh, which cnnot mke conventionl hydrogen ond to the cronyl, shows no shift in EC 50 in response to this muttion (Supplementry Tle 1). Log [EC 50 (mutnt)/ec 50 (wild type)] Log [EC 50 (mutnt)/ec 50 (wild type)] Muscle type ACh (WT) icotine (G153K) icotine (WT) Ction π energy (kcl mol 1 ) α4β2 ACh icotine ACh (µm) ppliction of ACh t concentrtions noted., Fit of dt in to the ill eqution. Error rs indicte s.e.m.; n This estlishes tht the ester muttion does not glolly lter the inding/gting chrcteristics of the receptor. The differentil ffinity of nicotine for 42 versus muscle-type receptors results from stronger interctions in the former with TrpB oth ction p nd hydrogen onding. Becuse the two receptors re identicl with regrd to the five residues tht mke up the romtic ox, fctor outside the ox must e influencing its precise geometry, such tht nicotine cn pproch TrpB more closely in 42 thn in muscle-type nachr. Pioneering work hs identified residues responsile for the fct tht 42 receptors show consistently higher ffinity thn the homopentmeric 7 neuronl receptors 29. At prticulr residue in loop B position 153, just four residues from TrpB muttions strongly influence ffinity. In high-ffinity 42 receptors this residue is Lys, nd this residue is proposed to help shpe the romtic ox y forming ckone hydrogen ond etween loops B nd C (Fig. 1). In the lower ffinity 7 neuronl receptor, residue 153 is Gly, nd moleculr dynmics simultions of 7 suggest tht Gly t 153 discourges the formtion of the hydrogen ond etween loops B nd C. Interestingly, the ligned residue in the muscle-type receptor is lso Gly, nd nturlly occurring G153S muttion is gin-of-function nd ssocited with congenitl mysthenic syndrome 30. We now report tht the muscle-type 1 G153K mutnt shows much higher ffinity for nicotine, nd tht, when this muttion is present, the ction p Wild type 4 pa 4 pa 200 ms 10 ms F 3 -TrpB Ction π energy (kcl mol 1 ) Figure 4 Fluorintion plots. ote tht in oth plots, ll dt sets shre the point t x kcl mol 21 (ction p energy for Trp); y 5 0 (lck circle). Moving to the left then corresponds to monofluoro-, difluoro-, trifluorond tetrfluoro-trpb. Ction p inding energies (x xes) re from ref. 9., Muscle-type receptor. The designtion WT indictes Gly t position 153., 42 receptor. 536 Figure 5 Single-chnnel recordings from wild-type 42 (conventionl expression) nd the F 3 -Trp mutnt (nonsense suppression) t site B, with nicotine pplied t EC 50 vlues (0.080 nd 1.2 mm, respectively). Lower trces re expnsions of the regions mrked y r in the upper trce. Records were otined in the cell-ttched configurtion with pipette potentil of 1100 mv nd re shown t 2 kz ndwidth. Chnnel openings re shown s downwrd deflections.

4 ATURE Vol Mrch 2009 interction to TrpB is strong. The dt re summrized in Supplementry Tle 1 nd Fig. 4. As expected, the ACh ction p interction is mintined in the muscle-type receptor with the G153K muttion. These dt indicte tht the loop B loop C hydrogen ond tht is nturlly present in 42 shpes the romtic ox so tht nicotine cn mke closer contct to TrpB, nd tht this structurl feture is sent or weker in the muscle-type receptor. Tken together, the present results indicte tht the higher ffinity of nicotine in the rin reltive to the neuromusculr junction is consequence of enhnced interctions with TrpB. A ction p interction tht is sent in the muscle-type receptor is quite strong in 42. In ddition, hydrogen ond to ckone cronyl tht is wek in the muscle type is enhnced in 42. Both effects re quite sustntil, nd in comintion they re more thn dequte to ccount fully for the differentil sensitivity to nicotine of the two receptors. The side chin of residue 153 in loop B distinguishes the two receptor types nd pprently influences the shpe of the inding site romtic ox, llowing stronger interction etween nicotine nd TrpB in high-ffinity receptors. METDS SUMMARY Whole-cell electrophysiologicl chrcteriztion of gonist-induced responses. Rt 42 nd mouse (1) 2 1cd ion chnnels were expressed in Xenopus levis oocytes. For 42 receptors, suunit stoichiometry ws controlled y vrying the 4:2 suunit rtio nd verified y voltge-jump experiments. Dose response mesurements for these chnnels were performed with holding potentil of 260 mv. Unnturl mino cid/-hydroxy cid incorportion. Unnturl mino cids nd -hydroxy cids were prepred, coupled to dca nd ligted to 74-mer TG73 s descried previously 15. Single-chnnel chrcteriztion of 42. Single-chnnel recording ws performed in the cell-ttched configurtion with pipette potentil of 1100 mv s descried previously 27. P open vlues were clculted from eventdetected dt using Clmpfit 9.2 single-chnnel serch. See ssocited ture Protocols pper y X.X. et l. (in the press). Full Methods nd ny ssocited references re ville in the online version of the pper t Received 26 ovemer 2008; ccepted 9 Jnury Pulished online 1 Mrch Romnelli, M.. et l. Centrl nicotinic receptors: structure, function, lignds, nd therpeutic potentil. ChemMedChem 2, (2007). 2. Corringer, P. J., Le overe,. & Chngeux, J. P. icotinic receptors t the mino cid level. Annu. Rev. Phrmcol. Toxicol. 40, (2000). 3. Coe, J. W. et l. Vrenicline: An 42 nicotinic receptor prtil gonist for smoking cesstion. J. Med. Chem. 48, (2005). 4. Gotti, C., Zoli, M. & Clementi, F. Brin nicotinic cetylcholine receptors: ntive sutypes nd their relevnce. Trends Phrmcol. Sci. 27, (2006). 5. Mnsvelder,. D., Keth, J. R. & McGehee, D. S. Synptic mechnisms underlie nicotine-induced excitility of rin rewrd res. euron 33, (2002). 6. shmi, R. et l. Chronic nicotine cell specificlly upregultes functionl 4* nicotinic receptors: sis for oth tolernce in midrin nd enhnced long-term potentition in perfornt pth. J. eurosci. 27, (2007). 7. Tpper, A. R. et l. icotine ctivtion of 4* receptors: sufficient for rewrd, tolernce, nd sensitiztion. Science 306, (2004). 8. Kurytov, A., Luo, J., Cooper, J. & Lindstrom, J. icotine cts s phrmcologicl chperone to up-regulte humn 42 cetylcholine receptors. Mol. Phrm. 68, (2005). 9. Zhong, W. et l. From initio quntum mechnics to moleculr neuroiology: A ction-p inding site in the nicotinic receptor. Proc. tl Acd. Sci. USA 95, (1998). 10. Brejc, K. et l. Crystl structure of n ACh-inding protein revels the ligndinding domin of nicotinic receptors. ture 411, (2001). 11. Sixm, T. K. & Smit, A. B. Acetylcholine inding protein (AChBP): secreted glil protein tht provides high-resolution model for the extrcellulr domin of pentmeric lignd-gted ion chnnels. Annu. Rev. Biophys. Biomol. Struct. 32, (2003). 12. Dougherty, D. A. Cys-loop neuroreceptors: structure to the rescue? Chem. Rev. 108, (2008). 13. Beene, D. L. et l. Ction-p interctions in lignd recognition y serotonergic (5 T 3A ) nd nicotinic cetylcholine receptors: The nomlous inding properties of nicotine. Biochemistry 41, (2002). 14. Dougherty, D. A. Physicl orgnic chemistry on the rin. J. rg. Chem. 73, (2008). 15. owk, M. W. et l. In vivo incorportion of unnturl mino cids into ion chnnels in Xenopus oocyte expression system. Methods Enzymol. 293, (1998). 16. Fonck, C. et l. ovel seizure phenotype nd sleep disruptions in knock-in mice with hypersensitive 4* nicotinic receptors. J. eurosci. 25, (2005). 17. Filtov, G.. & White, M. M. The role of conserved leucines in the M2 domin of the cetylcholine receptor in chnnel gting. Mol. Phrmcol. 48, (1995). 18. Lrc, C. et l. Chnnel gting governed symmetriclly y conserved leucine residues in the M2 domin of nicotinic receptors. ture 376, (1995). 19. Kerney, P. et l. Dose-response reltions for unnturl mino cids t the gonist inding site of the nicotinic cetylcholine receptor: Tests with novel side chins nd with severl gonists. Mol. Phrmcol. 50, (1996). 20. Moroni, M., Zwrt, R., Sher, E., Cssels, B. K. & Bermudez, I. 42 nicotinic receptors with high nd low cetylcholine sensitivity: phrmcology, stoichiometry, nd sensitivity to long-term exposure to nicotine. Mol. Phrmcol. 70, (2006). 21. elson, M. E., Kurytov, A., Choi, C.., Zhou, Y. & Lindstrom, J. Alternte stoichiometries of 42 nicotinic cetylcholine receptors. Mol. Phrmcol. 63, (2003). 22. Celie, P.. et l. icotine nd crmylcholine inding to nicotinic cetylcholine receptors s studied in AChBP crystl structures. euron 41, (2004). 23. nsen, S. B. et l. Structures of plysi AChBP complexes with nicotinic gonists nd ntgonists revel distinctive inding interfces nd conformtions. EMB J. 24, (2005). 24. Tlley, T. T. et l. Spectroscopic nlysis of enzylidene nseine complexes with cetylcholine inding proteins s models for lignd-nicotinic receptor interctions. Biochemistry 45, (2006). 25. Cshin, A. L., Petersson, E. J., Lester,. A. & Dougherty, D. A. Using physicl chemistry to differentite nicotinic from cholinergic gonists t the nicotinic cetylcholine receptor. J. Am. Chem. Soc. 127, (2005). 26. Deechongkit, S. et l. Context-dependent contriutions of ckone hydrogen onding to -sheet folding energetics. ture 430, (2004). 27. Englnd, P. M., Zhng, Y., Dougherty, D. A. & Lester,. A. Bckone muttions in trnsmemrne domins of lignd-gted ion chnnel: implictions for the mechnism of gting. Cell 96, (1999). 28. Koh, J. T., Cornish, V. W. & Schultz, P. G. An experimentl pproch to evluting the role of ckone interctions in proteins using unnturl mino cid mutgenesis. Biochemistry 36, (1997). 29. Grutter, T. et l. An -ond etween two residues from different loops of the cetylcholine inding site contriutes to the ctivtion mechnism of nicotinic receptors. EMB J. 22, (2003). 30. Sine, S. M. et l. Muttion of the cetylcholine receptor suunit cuses slowchnnel mysthenic syndrome y enhncing gonist inding ffinity. euron 15, (1995). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk B.. Cohen for dvice on single-chnnel recording nd nlysis. This work ws supported y the I (S 34407; S 11756) nd the Cliforni Tocco-Relted Disese Reserch Progrm of the University of Cliforni, grnt numer 16RT J.A.P.S. ws prtilly supported y n RSA trining grnt. Author Informtion Reprints nd permissions informtion is ville t Correspondence nd requests for mterils should e ddressed to D.A.D. (ddougherty@cltech.edu). 537

5 doi: /nture07768 METDS Whole-cell electrophysiologicl chrcteriztions of the gonist-induced responses. Rt 4 nd 2 mras s well s mouse 1, 1(L99S), c nd d mras were otined from oti lineriztions of the expression vector pamv, followed y in vitro trnscription using the mmessge mmchine T7 kit (Amion). The muttions for ech suunit were introduced ccording to the QuikChnge mutgenesis protocol (Strtgene). To express wild-type neuronl ion chnnels, 4L99A mra ws co-injected with 2 mra t vrious rtios (totl mra ng cell 21 ). Stge V VI Xenopus levis oocytes were injected nd incuted t 18 uc for h (whole-cell recording) or h (single-chnnel recording). Agonist-induced currents were recorded in two-electrode voltge clmp mode using the pusxpress 6000A (Moleculr Devices Axon Instruments) t holding potentil of 260 mv. Agonists were prepred in C 21 -free D96 solution nd pplied for 12 s followed y 2 min wsh with C 21 -free D96 solution etween ech gonist ppliction. Acetylcholine chloride nd (-)-nicotine trtrte were purchsed from Sigm/Aldrich/RBI. Dose response dt were otined for $6concentrtions of gonist nd for $5 oocytes. Mutnts with I mx of $100 na were defined s functionl. EC 50 vlues nd the ill coefficient were clculted y fitting the dose response reltion to the ill eqution. All dt re reported s men6 s.e.m. Voltge jump experiments were performed in the sence of ACh nd lso t EC 50 concentrtion of ACh. The memrne potentil ws held t 260 mv, nd stepped to 10 test potentils t 20-mV increments etween 170 mv nd 2110 mv for 400 ms ech. The voltge ws then held for 600 ms t 260 mv holding potentil etween ech episode. To isolte the ACh-induced currents, control trces ([ACh] 5 0) were sutrcted from the stedy-stte mplitudes of the ACh-induced currents of the test pulses. ormlized current voltge curves were generted using current mplitudes normlized to tht t 2110 mv. For ech 4L99A2 mutnt, normlized I 170 mv 6 s.e.m. from $5 cells ws reported. Unnturl mino cid/-hydroxy cid incorportion. itrovertryloxycronyl (VC) protected cynomethyl ester forms of unnturl mino cids nd -hydroxythreonine cynomethyl ester were synthesized, coupled to the dinucleotide dca, nd enzymticlly ligted to 74-mer TG73 tra CUA 15. The unnturl mino-cid-conjugted tra ws deprotected y photolysis immeditely efore co-injection with mra contining the UAG muttion t the site of interest. Approximtely ng mra nd 25 ng tra-mino cid or tra-hydroxy cid were injected into stge V VI oocytes in totl volume of 70 nl. For unnturl mino cid mutgenesis experiments in the muscle-type receptor, the 1, 1, c nd d suunits were co-injected in 10:1:1:1 rtio. All muscle-type receptors contined L99S muttion in the suunit. The fidelity of unnturl mino cid incorportion ws confirmed t ech site with wild-type recovery experiment nd red-through/reminocyltion test. In the wild-type recovery experiment, UAG mutnt mra ws co-injected with tra chrged with the mino cid tht is present t this site in the wild-type protein. Genertion of receptors tht were indistinguishle from the wild-type protein indicted tht the residue crried y the suppressor tra ws successfully nd exclusively integrted into the protein. In the red-through/ reminocyltion test, the UAG mutnt mra ws introduced with (1) no tra, (2) tra TG73 tht ws not chrged with ny mino cid or (3) tra TG73 enzymticlly ligted with dinucleotide dca. Lck of currents in these experiments vlidted the reliility of the nonsense suppression experiments. Single-chnnel chrcteriztion of 42. Single-chnnel recording ws performed in the cell-ttched configurtion on devitellinized Xenopus levis oocytes t uc with pipette potentil of 1100 mv, s descried previously 27. Pipettes were fricted from thick-wlled (inner dimeter mm, outer dimeter mm) KG-33 glss (Grner Glss Compny) nd coted with sylgrd (World Precision Instruments); they hd resistnces of MV. The th solution contined 120 mm KCl, 5 mm EPES, 1 mm MgCl 2 nd 2 mm CCl 2,p5 7.4, so tht the reversl potentil for gonist-induced currents of devitellinized oocytes ws,0 mv, nd the trnsmemrne potentil of the ptch ws,2100 mv. The pipette solution contined 100 mm KCl, 10 mm EPES, 1 mm MgCl 2,10mMK 2 EGTA, p nd ws supplemented with the indicted concentrtions of nicotine. Dt were collected using GeneClmp 500B mplifier (Axon Instruments) t full ndwidth (50 kz; 4-pole Bessel, 23 db) with CV GU hedstge. The signl ws then low-pss filtered (Avens Signl Equipment, AP220, 8-pole Bessel, 23 db 20 kz) nd smpled with Digidt 1320A nd Clmpex 9.2 (Axon Instruments) t 50 kz. nly ptches tht showed no simultneous ctivtions were nlysed. For ech mutnt, this ws $3 ptches from oocytes from two different donor frogs. Dt were filtered offline (Gussin, 23 db, 5 kz) nd electricl interference t hrmonics of 60 z ws removed if necessry. Event trnsitions were detected with Clmpfit 9.2 (single-chnnel serch). A ded time, t d, of 100 ms ws pplied to ll events. pen nd closed dwell time histogrms were generted s descried previously 31 nd fitted using the predefined log-trnsformed exponentil proility density function in Clmpfit 9.2. To delinete clusters, criticl closed durtion, t crit, ws defined using two seprte methods (Supplementry Discussion); in oth cses closed dwell times longer thn t crit were excluded from further nlysis. Sojourns to suconductnce stte (,85% of the full conductnce level) were treted s closed nd ccounted for,10% of the totl openings in ll records. The time-verge proility tht the chnnel is open (P open ) ws clculted s the totl open time divided y the sum of the revised totl closed time nd the totl open time. 31. McMnus,. B., Bltz, A. L. & Mgley, K. L. Smpling, log inning, fitting, nd plotting durtions of open-nd-shut intervls from single chnnels nd the effects of noise. Pflugers Archiv-Eur. J. Physiol. 410, (1987).