Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29The cation-p site in a nicotinic acetylcholine receptorUnnatural amino-acids define acetylcholine binding within 0.5 AngstromsSlide 321Bi 1 Lecture 7 Monday, April 10, 2006 The Central Dogma of Drugs and the Brain,Part 1:Drugs open and block ion channelsNCH3NNicotine2[neurotransmitter]openclosedchemical transmission atsynapses:electric fieldopenclosedelectrical transmission inaxons:actually, EMajor Roles for Ion Channelsfrom Lecture 63nicotineprocaine NCH3NOCH2H2CNH2CONH3CH2CCH2CH3botulinum toxinAtomic-scale Structures(Swiss-prot pdb viewer must be installed on your computer)NOHOHOCH3morphinemorphinehttp://www.its.caltech.edu/~lester/Bi-1/nicotine.pdbhttp://www.its.caltech.edu/~lester/Bi-1/procaine.pdbhttp://www.its.caltech.edu/~lester/Bi-1/morphine.pdbfrom Lecture 24nicotinic acetylcholine receptorMost drug receptors are membrane proteinsOutside the cellInside the cell = cytosol(view in ~1995)natural ligand(agonist)nicotine, another agonistMembrane = lipid bilayer~ 100 Å= 10 nmfrom Lecture 35 Overall topology of the nicotinic acetylcholine receptor(view in ~2000)outside the cell:5 subunitseach subunit has 4 -helices in the membrane (20 membrane helices total)Little Alberts figure 12-42© Garland publishingfrom Lecture 36 The acetylcholine binding protein (AChBP) from a snail, discovered in 2001, strongly resembles the binding region(Swiss-prot viewer must be installed on your computer)Color by chainShow 2 subunits,Chains,Ribbons5 subunitsLittle Alberts figure 12-42© Garland publishinghttp://www.its.caltech.edu/~lester/Bi-1/AChBP+Carb-5mer.pdbfrom Lecture 37http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdbThe AChBP binding site occupied by an acetylcholine analog (2004)http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdbfrom Lecture 38Binding regionMembrane regionCytosolicregionColored by secondary structureColored by subunit(chain)Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 106 from Lecture 39 How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 viewM2M1M3M4Ligand-bindingdomainfrom Lecture 310~60oclosedclosed-OHopenopen-OH-OH-OH-OH-OH5 kinked -helices rotate, removing the oily side chain ( = hydrophobic) from the poreand exposing side chains with -OH groups. This would provide a water-like (HOH) environment for the permeating ions.One hypothesis about the nature of nicotinic receptor gatingin the transmembrane domain11Twisting the knee, or straightening the leg? We don’t yet knowEngineering & Science, 200212A better way: record the current from channels directly?AFeynman’s ideafrom Lecture 6135 pA = 104 ions/ms20 msa nicotinic acetylcholine receptor exposed to acetylcholine Asensitive electronic ammeterdynamic range:5 s to 5 min 1 part in 108Implementing Feynman’s Idea14How ”tight” is the gigaohm seal? 1. Electrically tightSee next slideLittle Alberts 12-22A© Garland 1 m15How ”tight” is the gigaohm seal? 1. Electrically tightR =  l/AR ~ 109 = resistivity = 22 -cm;l = length = 10 m;A = area = 10 m x t (thickness);Therefore t ~ 2 x 10-11 m, or less than 1 Å!16acetylcholine in the pipette opens channels in the pipette2. Chemically tight The seal compartmentalizes molecules. Molecules outside the pipette do not mix with molecules inside the pipette acetylcholine outside the pipette opens channels outside the pipetteHow ”tight” is the gigaohm seal?17Alberts omitted all of the electronics!Asensitive electronic ammeterLittle Alberts 12-22D© Garland18Max DelbruckRichard FeynmanH. A. LCarver Mead“If you want to measure small, noisy signals, I have a Senior who can help”from Lecture 619Fred Sigworth ‘74 and Apostol’s Clockhttp://www.math.caltech.edu/classes/ma1c-An/index.html#textMa 1c-Analytic track, Spring 2006:http://www.info.med.yale.edu/bbs/faculty/sig_fr.htmlFred Sigworth’s Web page at Yale20Statistical analysis of single-molecule eventschannel opensnow we synchronize artificially on the opening eventn =1021Statistical analysis of single-molecule eventsn =1022from Chem 1b 2006 Lecture Series #5(Heath)23Concentration of acetylcholine at asynapseNumber of open channelsms0highclosedopenState 1 State 2k21all molecules begin here at t= 0units: s-124We wish to test a hypothesis that a particular side chain governs channel opening/closing rates~60oclosedclosed-OHopenopen-OH-OH-OH-OH-OH25Site-Directed Mutagenesis: the General IdeaRNAGene (DNA)measureHypothesis about an important side chain(s)Mutate the desired codon(s)“Express” theprotein with an altered side chain(s)26in vitro RNA synthesisRNA polymerase promoterDNAmeasureSite-Directed Mutagenesis on Ion ChannelsExpress by injecting into immature frog eggsMutate the desired codon(s)Latin, ‘in glass” measure27The identified side chain governs channel opening/closing ratesn =10n =10leucine (wild type)alanine28We wish to test a hypothesis that a particular side chain governs agonist binding29Measured “dose-response” relations verify that an identified side chain governs agonist-receptor interactionswild type (tryptophan)phenylalanineThe instrument (~ 90 MB!):http://www.moleculardevices.com/product_literature/download_form.php?docnum=475&prodid=108&useid=25&familyid=&interestid=30The cation- site in a nicotinic acetylcholine receptor We know that acetylcholine and nicotine bind within a “box” of aromatic amino acids; Electrophysiology agrees with crystallography! ~ 8 Angstromstryptophan31Unnatural amino-acids define acetylcholine binding within 0.5 Angstroms Quantum-mechanical calculations of cation- energyMeasured acetylcholine binding energyDougherty group, Caltech Chemistry32End of Lecture 7