Class Note for BIOC 462A at UA
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The Structure of the Potassium Channel Molecular Basis of K Conduction and Selectivity Declan A Doyle Joao Morais Cabral Richard A Pfuetzner Anling Kuo Jacqueline M Gulbis Steven L Cohen Brian T Chait Roderick MacKinnon The potassium channel from Sfrepfomyces lividans is an integral membrane protein with sequence similarity to all known Kquot channels particularly in the pore region Xray analysiswith data to 32 angstroms revealsthat four identical subunits create an inverted teepee or cone cradling the selectivity filter of the pore in its outer end The narrow selectivity filter is only 12 angstroms long whereas the remainder of the pore is wider and lined with hydrophobic amino acids A large waterfilled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer Main chain carbonyl oxygen atoms from the Kquot channel signature sequence line the selectivity filter which is held open by structural constraints to co ordinate Kquot ions but not smaller Naquot ions The selectivity filtercontainstwo Kquot ionsabout 75 angstroms apart This configuration promotes ion conduction by exploiting electro static repulsive forces to overcome attractive forces between Kquot ions and the selectivity filter The architecture ofthe pore establishes the physical principles underlying selective Kquot conduction Potassium ions diffuse rapidly across cell membranes through proteins called K4r channels This movement underlies many fundamental biological processes includ ing electrical signaling in the nervous sys tem Potassium channels use diverse mechanisms of gating the processes by which the pore opens and closes but they all exhibit very similar ion permeability characteristics All K channels show a selectivity sequence of K N Rb gt Cs whereas permeability for the smallest alkali metal ions Na and Li is immea surably low Potassium is at least 10000 times more permeant than Na a feature that is essential to the function of K channels Potassium channels also share a constellation of permeability characteris tics that is indicative of a multiion conduction mechanism The flux of ions in one direction shows highorder cou pling to flux in the opposite direction and ionic mixtures result in anomalous con duction behavior Because of these D A Doyle R A Pfuetzner A Kuo and R MacKinnon are in the Laboratory of Molecular Neurobiology and Bio physics and the Howard Hughes Medical lnstitute Rocky efeller University 1280 York Avenue New York NY 10021 USA J M Cabral and J M Gulbis are in the Laboratory of Molecular Neurobiology and Biophysics Rockefeller University 1280 York Avenue New York NY 10021 USA S L Cohen and B T Chait are in the Laboratory of Mass Spectrometry and Gaseous lon Chemistry Rockefeller University 1280 York Avenue NewYork NY 10021 USA To whom correspondence should be addressed Ermail mackinnrockvax rockefeller edu properties K channels are classified as long pore channels invoking the notion that multiple ions queue inside a long narrow pore in single file In addition the pores of all K channels can be blocked by tetraethylammonium TEA ions Molecular cloning and mutagenesis ex periments have reinforced the conclusion that all K channels have essentially the same pore constitution Without exception all contain a critical amino acid sequence the K channel signature sequence Muta tion of these amino acids disrupts the chan nels ability to discriminate between K and Na ions Two aspects of ion conduction by K channels have tantalized biophysicists for the past quarter century First what is the chemical basis of the impressive fidelity with which the channel distinguishes be tween K and Na ions which are feoature less spheres of Pauling radius 133 A and 095 A respectively Second how can K channels be so highly selective and at the same time apparently paradoxically exhib it a throughput rate approaching the diffu sion limit The 10 l margin by which K is selected over Na implies strong energetic interactions between K ions and the pore And yet strong energetic interactions seem incongruent with throughput rates up to 108 ions per second How can these two essential features of the K channel pore be reconciled Potassium Channel Architecture The amino acid sequence of the K chan nel from Streptomyces lividans KcsA K r channel 5 is similar to that of other K channels including vertebrate and inverte brate voltagedependent K4r channels ver tebrate inward rectifier and CaZTactivated K channels K channels from plants and bacteria and cyclic nucleotidegated cation channels Fig 1 On the basis of hydro phobicity analysis there are two closely related varieties of K channels those con taining two membranespanning segments per subunit and those containing six In all cases the functional K channel protein is a tetramer 6 typically of four identical subunits Subunits of the two mem branespanning variety appear to be short ened versions of their larger counterparts as if they simply lack the first four membrane spanning segments Although the KcsA K channel is a two membranespanning K channel its amino acid sequence is actually closer to those of eukaryotic six membrane spanning K4r channels In particular its sequence in the pore region located be tween the mernbranespanning stretches and containing the K channel signature sequence is nearly identical to that found in the Drosophila Shaker and vertebrate voltagegated K4r channels Fig 1 In an accompanying paper through a study of the KcsA K channel interaction with eukary otic K channel toxins we confirm that the KcsA pore structure is indeed very sim ilar to that of eukaryotic K channels and that its structure is maintained when it is removed from the membrane with deter gent 8 We have determined the KcsA K channel structure from residue position 23 to 119 by xray crystallography Table l The cytoplasmic carboxyl terminus resi dues 126 to 158 was removed in the prep aration and the remaining residues were disordered The KcsA K channel crystals are radiationsensitive and the diffraction pattern is anisotropic with reflections ob served oalong the best and worst directions at 25 A and 35 A Bragg spacings respec tively By data selection anisotropy correc tion introduction of heavy atom sites by sitedirected mutagenesis averaging and solvent attening an interpretable electron density map was calculated Fig 2 A through C This map was without main chain breaks and showed strong side chain density Fig 2C The model was refined with data to 32 A the data set was 93 complete to 302 A with 67 completeness between 33 A and 32 A maintaining highly restrained stereochemistry and keep ing tight noncrystallographic symmetry re straints The refinement procedure was wwwsciencernagorg 0 SCIENCE 0 VOL 280 3 APRIL 1998 69 monitored by minimizing the value Rfree 290 and its separation from Rcrystal lographic 280 The presence of four molecules subunits in the asymmetric unit of the crystal provides a very significant enhancement of the accuracy of the crys tallographic analysis first by enabling av eraging of the electron density over four crystallographically independent regions of the multiple isomorphous replacement MIR map and second by providing a powerful set of constraints on the atomic model during refinement The K channel is a tetramer with four fold symmetry about a central pore Fig 3 A and B Like several other membrane proteins it has two layers of aromatic amino acids positioned to extend into the lipid bilayer presumably near the membranewa ter interfaces Fig 3C 10 Each subunit has two transmembrane OLhelices connect ed by the roughly 30 amino acid pore re gion which consists of the turret pore he lix and selectivity filter Fig 3 A and B A subunit is inserted into the tetramer such that one transmembrane helix inner helix faces the central pore while the other outer helix faces the lipid membrane The inner helices are tilted with respect to the mem brane normal by about 25 and are slightly kinked so that the subunits open like the petals of a flower facing the outside of the cell The open petals house the structure formed by the pore region near the extra cellular surface of the membrane This re gion contains the KTr channel signature sequence which forms the selectivity filter 4 The essential features of subunit pack ing can be appreciated by viewing the rela tion between the four inner helices and the four pore helices Fig 3D The four inner helices pack against each other as a bundle near the intracellular aspect of the mem brane giving the appearance of an inverted teepee The pore helices are slotted in be tween the poles of the teepee and are di rected with an aminotocarboxyl sense toward a point near the center of the chan nel Fig 3 A B and D This pore helix arrangement provides many of the intersub unit contacts that hold the tetramer togeth er and as discussed below is also critical in the operation of the ion conduction pore Sequence conservation among KTr chan nels including ones with two and six mem branespanning segments as well as cyclic nucleotidegated cation channels is stron gest for the amino acids corresponding to I PORE REGION I OUTER H ELIX FORE HELIX INN ER H ELIX kcsa kch SLMTAPYPSIETM DIV c105 SLGNALWWSPVTI DIS shake SIPDAPWWAWTM DMT hKvl 1 SIPDAPWWAWSMT DMY hDRK SIPASPWWATITM DIY Parana QYLHSLYWSIITM DIT Celegans SIPLGLWWAICTM DMT InSlo TYWECVYLLMVTM DVY caliac NPLGAMWLISITP DMv AKTl RWTSMYWSITTL DLH harg KWTALYPTPSSL I 5 rank GMTSAPLPSLETQ hgirk GPVSAPLPSIETE olCNG EYIYCLYWSTLTLT ETPP rodCNG KWYSLYWSTLTLT ETPP PvTLWGRLvAWv39MvIx PVSESARLPTISVIISG PSTPPGRVIASILMLIG PVGPWGKIVGSLCVIAG PVTIGGKIVGSLCAIAG PKTLLGKIVGGLCCIAG PQNLRERVPAVGMALS PHTSPGRLVGSLCAV39MG AKTTLGRLPMVPPILGG PHTYCGKGVCLLTGIMG PV39NTKEMIPDIPY39MLP PNTNSEKIPSICV MLIG PRPVTEQCATAIPLLIPQSILG YRVITDKCPEGIILLLIQSVLG PVKDEEYLPVIPDPLIG PVRDSEWPVWDPLIG Fig 1 Sequence allgnment of selected Kt channels and cycllc nucleotldeigated cnannels Tne numberan and secondary structural elementstor abovetne sequences Selectlvttytllter red llnlng o tne Streptomyces Vdans Kt cnannel KcsA ls glven ttne caVlty and lnner pore blue resldues ln wnlcn the nature of the slde cnaln ls preserved gt50 slmllanty grey Tne sequences are KcsA Streptomyces Vdans accesslon number acc PlR S60t 72 lltcn Eschercna C0 acc GenBanllt U24203 clost Costrdum acetobutylcum Genome Tnerapeutlcs Corp Shaker Drosopnla meanogaster acc PlR 800479 nKvt t Homo sapens acc Swtssprot Q09470 nDRK H sapens acc PlR S3t76t Parame ParameCum tetraaurela acc GenBanllt Ut9908 Celegans Caenornabdts eegans acc GenBanllt AF005246 mSlo Mus muscuus acc PlR A48206 caliact H sapens acc GenBanllt AFO3t8t5 AKTt Arabdopss thelane acc PlR 862694 nerg H sapens acc PlR l38465 romllt Flattus nonegcus acc Swtssprot P35560 nglrk ens acc Swtssprot Qt 6280 rodONG H sapens H sapens acc GenBanllt 878684 olONG H sap7 acc PlR A42t 6t Tne last two sequences separate from the rest are from cycllc nucleotldergated cnannels wnlcn are not Kt selectlve 70 the pore region and the inner helix Even Na and Ca2 channels show distant relat edness over these segments The teepee ar chitecture of the KTr channel pore likely will be a general feature of all of these cation channels with four inner helices arranged like the poles of a teepee four pore helices and a selectivity filterituned to select the appropriate cationilocated close to the extracellular surface This structure of the KcsA KTr channel is in excellent agreement with results from functional and mutagenesis studies on Shake er and other eukaryotic K channels Fig 4 The poreregion of KTr channels was first defined with poreblocking scorpion toxins l l These inhibitors interact with amino acids Fig 4 white comprising the broad extracellularfacing entryway to the pore 12 The impermeant organic cation TEA blocks KTr channels from both sides of the membrane at distinct sites 13 Amino acids interacting with externally and inter nally applied TEA are located just external to Fig 4 yellow and internal to Fig 4 mustard the structure formed by the signa ture sequence amino acids l4 l5 Alter ation of the signature sequence amino acids Fig 4 red main chain atoms disrupts KTr selectivity Amino acids close to the intracellular opening on the Shaker KTr channel map to the inner helix on the KcsA KTr channel 16 Interestingly expo sure to the cytoplasm of the region above the inner helix bundle Fig 4 pink side chains requires an open voltagedependent gate whereas the region at or below the bundle Fig 4 green side chains is exposed whether or not the gate was open The correlation between the transition zone for gatedependent exposure to the cytoplasm in the Shaker KTr channel and the inner helix bundle in this structure has plications for mechanisms of gating in K channels im General Properties of the Ion Conduction Pore As might have been anticipated for a cation channel both the intracellular and extra cellular entryways are negatively charged by acidic amino acids Fig 5A red an effect that would raise the local concentration of cations while lowering the concentration of anions The overall length of the pore is 45 A and its diameter varies along its distance Fig 5B From inside the cell bottom the pore begins as a tunnel 18 A in length the internal pore and then opens into a wide cavity NlO A across near the middle of the membrane A K ion could move throughout the internal pore and cavity and still remain mostly hydrated In contrast the selectivity filter separating the cavity SCIENCE 0 VOL 280 3 APRIL 1998 0 wwwsciencemagorg RESEARCH ARTICLES from the extracellular solution is so narrow that a K ion would have to shed its hy drating waters to enter The chemical com position of the wall lining the internal pore and cavity is predominantly hydrophobic Fig 5A yellow The selectivity filter on the other hand is lined exclusively by polar main chain atoms belonging to the signa ture sequence amino acids The distinct mechanisms operating in the cavity and internal pore versus the selectivity filter will be discussed below but first we introduce the determination of K ion positions in the pore Potassium channels exclude the smaller alkali metal cations Li radius 060 A and Na 095 A but allow permeation of the larger members pf the series Rb 148 A and Cs 169 A In fact Rb is nearly a perfect K 133 A analog because its size and permeability characteristics are very similar to those of K Because they are more electron dense than KT Rb and Cs allow visualization of the locations of per meant ions in the pore By difference elec tron density maps calculated with data from crystals transferred into Rb containing Fig 6a or Cs containing Fig 6b solu tions multiple ions are well defined in the pore The selectivity filter contains two ions inner and outer ioons located at op posite ends about 75 A apart center to center In the Rb difference map there actually are two partially separated peaks at the inner aspect of the selectivity filter These peaks are too close to each other 26 A to represent two simultaneously occu pied ion binding sites We suspect that they represent a single ion on average in rapid equilibrium between adjacent sites The single inner ion peak in the Cs difference map undoubtedly reflects the lower resolu tion at which the map owas calculated to 5 A for Cs versus 40 A for Rb because the Rb difference map when calculated at the same lower resolution also shows only a single peak at the Cs position The Rb positions correspond to strong peaks pre EXPERIMENTAL MAPS EXPERIMENTAL MAPS Fig 2 Experimental electron density map Ste reoviews of the experimental electron density map contoured at 1 039 covering nearly an entire subunit removed from the tetramer of the final model The map was calculated at 82 A resolu tion with the following Fourier coefficients native sharpened amplitudes and MIR solvent flattened averaged phases A Foreground map showing inner helix loop structures and selectivity filter background the pore helix and outer helix CPK spheres show positions of mercury atoms used as residue markers from the top marked residues are Leu86 Leu9O and Val93 B Alternative view Foreground pore helix and part of outer helix background selectivity filter and turret CPK sphere marks position of Ala C Close up view of electron density wwwsciencemagorg 0 SCIENCE 0 VOL 280 3 APRIL 1998 71 sumably K4r ions in a high contour native electron density map not shown Thus the selectivity filter contains two K4r ions A third weaker peak is located below the selectivity filter at the center of the large cavity in the Rb difference map Fig 6a cavity ion and in the Cs difference map at a lower contour not shown Electron density at the cavity center is prominent in lower diffuse peak The difference electron density maps show this to be related to the presence of one or more poprly localized cations situated at least 4 A away from the closest protein groups The Cavity and Internal Pore Why is there a 10 A diameter cavity in the center of the channel with an ion in it Fig 5B and Fig 67 Electrostatic calcula tions show that when an ion is moved along a narrow pore through a membrane it must cross an energy barrier that is maximum at the membrane center 17 The electrostatic field emanating from a cation polarizes its environment bringing the negative ends of dipoles closer to it and thereby stabilizing it At the bilayer MIR maps even prior to averaging Fig 6c Table 1 Summary of data pollecthh ahd reflhemehtastatlstlcs 0rystals space group02 a 128 8Ab 689A c 1120A B 124 6 were flashefrozeh by belhg trahsferred dlrectly from the crystal mother llduorto a stream of bOlledVOff hltrogeh 24 Because crystals of the mutaht L900 dlffracted slghlflcahtly better thah Wlldetype protelh crystals the former were used for hatlve data collecthh Data were collected from multlple crystals ahd SlX sets were selected ahd merged to form the hatlve data set used for structure determlhathh Mercury derlvatlves were obtalhed by dlrect addlthh of methyl mercury to the crystalllzathh soluthh of cystelhe mutaht crystals MALDleTOF mass spectrometry cOhflrmed 60 to 90 derlvatlzathh of crys tals prlor to data collecthh All data were collected at 00mell ngh Ehergy SyhchrotrOh Source CHESS stathh A1 Wlth the PrlhcetOh 2K 00D 25 Data were processed Wlth DENZO ahd SOALEPAOK 26 ahd the 00P4 paclltage 27 Heavy atom poslthhs were determlhed Wlth SHELX797 28 ahd crossedlfferehce Fourler ahalysls These poslthhs cOhflrmed thefourfold hOhcrystallographlc symmetry observed lh the selferotathh PattersOh fuhce thh ahd allowed the determlhathh of lhltlal orlehtathh matrlces Ah lhltlal model 90 complete was bullt lhto a solveht flattehed 64 solveht 000 teht fourfold averaged electrOh dehslty map Wlth the program 0 29 The traclhg of the model was facllltated bythe use of the mercury atom poslthhs as resldue marllters L860 was used solely for thls purpose After torlehal center the polarizability of the surround reflhemeht Wlth strlct fourfold hOhcrystallographlc symmetry cOhstralhts Wlth XAPLOR 3 851 3 thls model was used lh the ahlsotroplc scallhg sharpehlhg 31 of the hatlve data Wlth XAPLOR The structure factor slgma values were also rescaled approprlately ahd the corrected data were used for all subsequeht procedures Fourfold averaglhg solveht flattehlhg ahd phase extehleh were applled lh Dlvl 32 resultlhg lh a marked lmprovemeht of the electrOh dehsltythat allowed correcthh of the model ahdthe bulldlhg of addlthhal resldues Reflhemeht cOhslsted of rouhds of poslthhal lh the lhltlal stages phase lhformathh was also lhcluded as a restralht ahd grouped Befactor reflhemeht lh XAPLOR Fourfold hOhcrystallographlc symmetry was hlghly restaralhed Wlth the force cOhstaht for poslthhal restralhts set as 1000 lltcalmolA2 The dlffuse th cloud descrlbed lh the text was lhltlally modeled as Ohe or more K ths ahd several water molecules however the results were uhsatlsfactory Therefore thls ahd other strOhg uhmodelled dehslty preseht lh solvehteflattehed maps ho averaglhg lhcluded was Fourler bacllte trahsformed scaled ahd lhcluded lh the reflhemeht procedure as partlal structure factors The flhal model lhcludes amlho aclds 23 to 119 of each chalh Thefollowmg resldues weretruhcated Arg27 to 013 He60 to 07 Arg6A to 013 Glu7 to 013 ahd Arg 7 to Na The stereochemlstry ls strOhgly restralhed Wlth ho outllers Oh the Ramachahdrah plot The hlgh Befactor values reflect the lhtehslty decay of the data beyOhd 4 A Data coect0rl and phasrlg Resoluthh compleleness Phaslhg Data set a Reduhdahcy overallouter Fl R70ullls1l A 0 merge power r L9007a 15 073 7 3 5 91 393 3 0 071 1 61 0 70 L9007b 15 073 7 7 0 91 5941 0 083 1 87 0 50 V930 15 073 7 41 98 3991 0 075 1 35 0 63 A320 15 074 0 2 3 84183 8 0 076 1 45 0 66 A290 15 075 0 2 7 73 974 0 0 063 1 03 0 85 A420 15 076 5 2 0 90 790 3 0 057 0 97 0 81 L860 30 076 0 2 3 58 758 9 0 057 7 7 039 of measured data Wlth 039 gt 2 Natlve 30 073 2 61 93 3 0 086 15 8 75 Outer Shell 3 373 2 2 3 66 6 0 286 3 9 50 Arlsotropc correct0n Average F Oalvlll Average P Q Mll 30 0732A 3 4732A Before sharpehlhgql 0 76 0 55 After sharpehlhqu 0 83 0 64 Re nement a Rootemearlesquare deVatorl of Resoluthh 10 073 2 A BOhd ahgles 1 096 a Recryst 28 0 BOhd lehgths 0 005 A Refree 29 0 Ncs related atoms 0 006 A No of reflecthhs Wlth lFlUl Fl gt 2 12054 Befactorfor hcs related atoms 10 A2 No of protelh atoms 710 per subuhlt Befactorfor Homebo ded atoms 36 A2 No of llgahd atoms Meah Befactorfor malhechalh atoms Meah Befactor for sldeechalh atoms 90Af 110A2 1 water 3 Kquot ths RWW 22 IE TPhaslhg power IFH I E IFerullls Elleh Fpl thollE IFpH Fpl only for centrlc data same as Flrcryst but calculated on 10 of data selected lrl thlh resolutloh shells and excluded from reflhemeht Flrcryst EleeFmoabdEIFDL Flrfree the llFlgure of merlt cllll39l both cases fourfold averaglrlg and solvent flattehlhg were applled ls the observed lrlterlslty ls the average lrlterlslty FH ls the rootrmeahrsduare heavyratom structure factor E lsthe lack of closure error FpH lsthe structure factorforthe derlvatlve F lsthe structure factorfor the hatlve FHo ls the calculated structure factor forthe heavy atom and Fpmm lsthe calculated hatlve structure factor 72 SCIENCE 0 VOL 280 3 APRIL 1998 0 wwwsciencemagorg ing medium is minimal and therefore the energy of the cation is highest Thus sim ple electrostatic considerations allow us to SELECTIVITY FILTER iNNEFi HELICES understand the functional significance of the cavity and its strategic location The cavity overcomes the electrostatic desta TUHRET SELECTIVITY FILTER HELICES RESEARCH ARTICLES bilization resulting from the low dielectric bilayer by simply surrounding an ion with polarizable water A second feature of the K channel structure also stabilizes a cat ion at the bilayer center The four pore helices point directly at the center of the cavity Fig 3 A B and D The amino to carboxyl orientation of these helices will impose a negative electrostatic cation at tractive potential via the helix dipole effect 18 The ends of the helices are rather far 8 A from the cavity center but all four contribute to the effect Therefore two properties of the structure the aqueous cavity and the oriented heli ces help to solve a fundamental physical problem in biology how to lower the electrostatic barrier facing a cation cross ing a lipid bilayer Thus the diffuse elec tron density in the cavity center Fig 6C likely reflects a hydrated cation cloud rather than an ion binding site Fig 7 Alternatively the channel could have overcome the destabilizing electrostatic effects of the bilayer center by lining the entire pore with a polarizable surface put ting ion binding sites along its entire length But the structure shows that with the exception of the selectivity filter the pore lining is mainly hydrophobic a gen eral property of K channels Fig 1 This conclusion was anticipated by the land marllt experiments of Armstrong which showed that hydrophobic cations bind in lt INTRACELLULAFI Fig 3 Views of the tetramer A Stereoview of a ribbon representation illustrating the three dimen sional fold of the KcsA tetramer viewed from the extracellular side The four subunits are distin guished by color B Stereoview from another perspective perpendicular to that in A C Rib bon representation of the tetramer as an integral membrane protein Aromatic amino acids on the membrane facing surface are displayed in black D Inverted teepee architecture of the tetramer These diagrams were prepared with MOLSORIPT and RASTER SD 33 wwwsciencemagorg 0 SCIENCE 0 VOL 280 3 APRIL 1998 73 the pore of KTr channels What is the significance of the hydrophobic lining We suggest that it would be counterpro ductive to achieving a high throughput of K ions were the lining of the channel to interact strongly with ions outside of the selectivity filter The hydrophobic lin ing presents a relatively inert surface to a diffusing ion over most of the length of the pore In summary the inner pore and cavity lower electrostatic barriers without creat ing deep energy wells The structural and chemical design of this part of the pore ensure a low resistance pathway from the cytoplasm to the selectivity filter facili f 059 I60 656 i H vpz 4 A57 V84 J 55 RM L31 r H m 5 t l 5 red GYG main chain only are absolutely required tating a high throughput Functional ex periments on K channels support this conclusion When TEA from the cyto plasm migrates to its binding site at the top of the cavity gt50 of the physical distance across the membrane Figs 4 and 5 it traverses only about 20 of the transmembrane voltage difference 15 Thus 80 of the transmembrane voltage is imposed across the relatively short se lectivity filter The rate limiting steps for a K ion traversing the channel are thereby limited to this short distance In effect the K channel has thinned the relevant transmembrane diffusion distance to a mere 12 A The Selectivity Filter The atomic model for the K channel selectivity filter was based on the experi mental electron density map which showed a continuous ridge of electron density attributable to the main chain as well as strong Val and Tyr side chain density directed away from the pore Fig 8A We also used KTr ion positions de fined by difference Fourier analysis Figs 6 and 8A yellow density and our knowl edge of alkali metal cation coordination in small molecules The side chain locations preclude their direct participation in ion coordination leaving this function to the for Kt selectivity 4 This figure was prepared with MOLSCRIPT and RAS TERBD Fig 5 right Molecular surface of KcsA and contour of the pore A A cutaway stereoview displaying the solventaccessible surface of the Kt channel colored according to physical properties Electrostatic potential was calculated with the program GRASP assuming an ionic strength equivalent to 150 mM KCI and dielectric constants of 2 and 80 for protein and solvent respectively Side chains of Lys Arg Glu and Asp residues were assigned single positive or negative charges as appropriate and the surface coloration varies smoothly from blue in areas of high positive charge through white to red in negatively charged regions The yellow areas of the surface are colored according to carbon atoms of the hydrophobic or partly so side chains of several semiconserved residues in the inner vestibule Thr75 lle OO Phe 03 Thr 07 Alalog Ala Valll5 The green CPK spheres represent Kt ion positions in the conduction pathway B Stereoview of the entire internal pore Within a stick model of the channel structure is a threedimensional representation ofthe minimum radial distance from the center of the channel pore to the nearest van der Waals protein contact The display was created with the program HOLE 34 74 SCIENCE VOL 280 3 APRIL 1998 wwwsciencemagorg maln chaln atoms The pleclse ollentatlon of lndlyldual cathonyl oxygens cannot be dlscemed at the lesolutlon of thls xelay analysis but we plopose that they ale dllected lnwald to account fol Kquot lon cooldlnatlon Flg 8B A slngle watel molecule the only one modeled ln the stluctule located between the two Kquot lons ln the selectlylty flltel was lustlfled by the plesence of a stlong electlon dene slty peak ln the expellmental map whlch was neyel assoclated wlth an lon peak ln the dlffelence Foullel maps l9 The stluctule of the selectlylty flltel exe hlblts two essentlal featules FIISt the maln chaln atoms cleate a stack of sequentlal oxygen llngs and thus affold numelous closely spaced sltes of sultable dlcnenslons tot cooldlnatlng a dehydtated llt lon The llt lon thus has only a yety small dlstance to dlffuse flom one slte to the next wlthln the selectlylty flltel The second lmpoltant stluctulal featule of the selectlylty flltel ls the ploteln packlng alound lt The Val and Tyl slde chalns flom the VeGeYeG se quence polnt away flom the pole and make Fig 6 ldentlllcatlon of permeant lon posltlons ln the pore aARb dlfn terence Fourler map cal culated to 40 A and contoured at o o ldentle lles two strong peaks correspondlng to lons ln the selectmty nlter lnner and outer lons and a weaker peak corree spondlng to lons ln the cavltycavlty lon The ll ln ner lon denslty has two closely spaced peaks bACsdlnerenceFoue her map calculated to OUTER ION INNER ION CAVITY ION speclflc lntelactlons wlth amlno aclds flom the tllted pole hellx Togethel wlth the pole hellx Ttp lesldues the foul Tyt slde chalns folm a masslye sheet of alomatlc amlno acids twelye ln total that ls posle tloned llke a cuff alound the selectlylty flltel Flg ac The hydlogen bondlngy tot example between the Tyt hydloxyls and Tlp nltlogensy and the extenslye yan del Waals contacts wlthln the sheet offel the lmmedlate lmplesslon that thls stluctule hehayes llke a layet of spllngs sttetched ladlally outwald to hold the pole open at lts ptopet dlametet How does the Kquot channel stluctule account fol lts plodlglous lon selectlye plopeltles When an lon entels the selece tlylty flltel lt eyldently dehydlates neale ly completely To compensate fol the enelgetlc cost of dehydtatlon the calbone yl oxygen atoms must take the place of the watel oxygen atoms come ln yely close contact wlth the lon and act llke sulloe gate watel 20 21 The stluctule leyeals that the selectlylty flltel ls held open as lf to pleyent lt flom accommodatlng a Naquot 6 b C ll 5 0A and contoured at o a shows the lnner and outer lon peaks ln the selecllvlty lllter Both dlfferel39lce Fourler maps were calculated wlth Fourler coemclents Fsoallt e Flnatlveeunsharpened and MR phases 0 Electron densltymap contoured at l o showlng dltluse denslty at the caVltylon poslllon Thls map was calculated wlth the followlng Fourler coemoents unsharpened natlve amplltudes and MR solventllattened phases no averaglng lntormatlon was lncluded Fig 7 Two mechanlsms by whlch the k channel stablllzes a catlon ln the mlddle of the membrane Flrst a large aque ous cale stablllzes an lon green ln the otherwlse hydro phoblc membrane lntenor Sec ond onented hellces polnt thelr partlal negatlve charge carboxe yl end red towards the caVlty where a catlon ls located www5clencemagolg 39 SCIWCE 39 VOL 280 39 3 APRIL 1998 RES AR TI LES lon wlth lts smallel ladlus We plopose that a Kquot lon flts ln the flltel pleclsely so that the enelgetlc costs and galns ale well balanced The stluctule of the selectlylty flltel wlth lts moleculal spllngs holdlng lt open pleyents the calbonyl oxygen atoms flom apploachlng close enough to come pensate fol the cost of dehydlatlon of a Naquot lon In about 150 mM K the selectlylty flltel contalns two Kquot lons Flgs 6 and 8 The lons ale located at opposlte ends of the selectlylty flltel sepalated by about 75 A loughly the avelage dlstance hetween llt lons ln a 4 M KCl Solution and ln the selectlylty flltel thele ale no lntelyenlng Cl anlons to balance the chalge We thelefole conclude that the selectlylty flltel attlacts and concentlates Kquot lons But how does such a selectlylty flltel eyel conduct lons The stluctule lmplles that a slngle Kquot lon would be held yely tlghtlyy but that the plesence of two Kquot lons lesults ln mutual epulslony hence thell locatlons neal opposlte ends of the selectlylty flltel Thus when a second lon entetsy the at tlactlye folce between a Kquot lon and the selectlylty flltel becomes pelfectly bale anced hy the lepulslve folce between lons and thls ls what allows conductlon to oce cul Thls plctule accounts fol both a stlong lntelactlon between Kquot lons and the selectlylty flltel and a hlgh thloughput medlated by electlostatlc lepulslon On the basls of functlonal measulementsy the same concept of destablllzatlon by multle ple lon occupancy has been ptoposed tot Ca channels 22 and fol Kquot channels 23 and may be a genelal ptopetty ofall selectlye lon channels Summary We plopose that the followlng pllnclples undellle the stluctule and opelatlon ofKquot channels l The pole ls constlucted of an lnyelted teepeey wlth the selectlylty flltel held at lts wlde end Thls alchltectule also descnhes the pole of cycllc nucleotldee gated channels and ptohahly Na and Ca channels as well In The nallow selectlylty flltel ls only 12 A long wheteas the lemalndel of the pole ls wldel and has a telatlyely melt hydtophohlc llnlng These stluctulal and chemlcal plopeltles fayol a hlgh Kquot thloughput by mlnlmlzlng the dlstance oyel whlch Kquot lntelacts stlongly Wlth the channel m A lalge watelefllled caylty and hellx dlpoles help to oyelcome the hlgh electlostatlc enelgy balllel faclng a catlon ln the low dlelectllc memblane centel ly The Kquot selectlylty flltel ls llned hy cathonyl oxygen atoms whlch ptoylde multlple closely spaced sltes The flltel ls constlalned ln an optle 75 mal geometty so that a dehydtated Kquot Ion ftts wtth ptopet cootdmatton but the Naquot Ion 15 too small v Two Kquot tons at close pIOXImItV m the selecttvtty ftltet tepel each othet The tepulston ovetcomes the otherwtse sttong tntetactton between Ion and plotem and allows apld conductton In the settmg of htgh selecttvtty Fig 3 Detatted mews ot the w channet seteca ttytty tttter A stereoytew ot the expertmenta etectron denstty green tn the setectMty t39ttter The map was cacutated wtth nattveasharpa ened amptttudes and MtRasotventa atteneda ayeraged phases The setecttytty t39ttter ot three subuntts ts shown as a SUCK representatton wtth seyerat stgnature sequence restdues tan beted The Rb dttterence map yettow ts atso shown B stereoytew ot the setecttytty t39ttter tn a stmttar ortentatton to A wtth the chatn ctosest to the vtewer removed The three chatns reprea sented are comprtsed ot the stgnature sea ouence amtno adds Tm Vat tht Tyn ety Tuna ntng trorn bottom to top as tabeted tn stngtea tetter code The va and Tyr stde chatns are dtrected away trorn the ton conductton paths Way whtch ts ttned by the man chan carbonyt oxygen atoms Two K tons green are tocated atoppostte ends otthe setecttvtty tten roughty wso W was W37 w v76 v18 V13 was wa7 67 was REFERENCES AND NOTES 1 B HtHe lama Channets afExcttabe Membranes 17 nauen Sundertand MA ed 2 1992 2 A L Hotghtn andR 0 KeynesJ Rhystet Luna0n 129 61 1955s Hagrwaras Mtyazakh s Krasne 5 01mm Gen Rhystet 70 269 1977R Httteand W Schwanz tbrd 72 409 1976u Neytonandc MtHen tbd 92 549 1966 3 c M Armstrong andL BtnstocK J Gen Physto 4st 659 1965 c M Armstrorg tbd 50 491 1966tbd 54 553 1969thd 59 4131971 4 L HegtnbothamT Abramwth MacKmnomSm me 253 1152 1992 L Hegtnbo hamZ LthT Abramwrh R MacKmnom J Btophys est 1061 1994 5 H Schemptetah EMBOJ 14 5170 1995 L Hegtnbothan E Odessem c MtHen Btechemtszry 36 10335 1997 0 Marten Conesand E Reroon bup 10343 R MacKmnorh Name 350 232 1991 Cenam K ohannets oontatn the equtyaent ot two subuntts tn a stngte open readtng trame These are thought totormthetetramerthroughtheassembtyot two dtmersubunnsM A Ketchumetat Namrei ii 690 1995 6 R MacKtnnon eta Some 290 106 1996 9 G u KleywegtandR u ReacLSmcIure 5 1557 1996 10 u DetsenhoferetaNature 319 6161965 5 W Cowanmatbtd 353 727 1992A Kreusch and G E SchutLJ M0 Stat 243 691 1994 11 R MacKtnnon and c MtHen shtertce 245 1362 1969 12 R MacKtnnom L Hegtnbo hamT AbramwnJVar run 5 767 1990 M Stockerand c MtHen Pres Nat Acad Sat USA 919509 1994 s A N Gotds etru 0 u pheasant 0 when Neuron 121 1377 1994gt thagoandR MacKtnnomSctance 268 307 1995 u AtyaretaNeumn 15 1169 19950 Naranpandc MtHembtd 161230996 R Ranganathamd H LewtsR MacKmnon tbrd p 131A GrossandR MacKmnormbmp 399 13 c M Armstrong and B HtHeJ Gen Phystn 59 366 1972 14 R MacKmnon and G VeHerh Spence 250 276 1990 15 G Vehem M E JurmarhT Abramwrh R MacKmn nomth 2511939 1991 16 v Ltut M Hotrrgrerh M E Jurmarh G VeHerhNar mnl9 75 1997 17 V A Parsegtarh Ann Nv Acad Sat 264 161 1975 16 0 Sat M BycromA R FersmVaxure 335 740 1966 u Aqvtsh H Luecka E A Qumhq A Narshet Pres Nat Acad Sat USA 99 2026 1991 0 u Lockhan and P s Ktmtsct nce 257 947 1992 tbtd 260 196 1993 19 The temperature taotors tor Vat7E and Sty ntatn ohatn atoms but not stde ohatn atoms re ned to hgheryaues than tor netghbortng atoms Thts resutt can be expLatned bythe dttterenoe Fourteranatyst whtoh shows aternattye postttons otthe tnnerK on tn the seteettyty tttter and theretora by tnterence attemattye contorrnattons ot the coordtnaltng rrtatn ohatn atoms dependtng on the toeaton ot the w m on 20 E Bezantha and c M ArmstrongJ Gen Physto 60 566 1972 21 B HTHembrd 6L669 1973 22 W Atmers and E W McCLesKeyJ Rhystet Luna den 353 565 1964 P Hess and R W Tsterh Name 309 453 1964 7 5Aapart wtthastngtewatermotecute red tn between The tnner ton ts deptcted as tn raptd eoutttbrtum 23 New and c Mtter J Gm Phystg 92 569 between adacent coordtnatton sttes The t39ttter ts surrounded by tnner and pore hettces whtte Atthough 1966 not shown the modet accounts tor hydrogen bondtng otatt amtde nttrogen atoms tn the setectMty t39ttter 24 The K096 gene was subc oned We pOEeO Owen except tor that otety77 0 Asectton ot the mode perpendtcutar to the pore at the teyet ot the setecttytty g t39ttter and wewed trorn the cytoptasm The ytew htghttghts the network ot aromattc amtno adds surround Omen 7 er We med New was ex mg the setecttytty t39ttter Tyrostnea78 trorn the setectMty t39ttter W8 tnteracts through hydrogen bondtng traeted by homogentzatton and sotubtttzaton tn 40 and van der Waats contacts wtth two Trp restdues WBZ W58 trorn the pore hettx mM deeytmatostde Anatraoe The Kcya ehannet 76 SCIWCE 39 VOL 280 39 3APRIL 1998 39 www501encemag01g was purified on a cobalt affinity column Thirtyrfive carboxyl terminal amino acids were cleaved by chye motrypsin proteolysis The truncated channel was purified to homogeneity by gel filtration and the der tergent exchanged in a final dialysis step against 5 mM NNrdimethyldodecylaminerNroxide LDAO Crystals were grown at 20 C with the sitting drop method by mixing equal volumes of protein solution 5 to 10 mgml 150 mM KCl 50 mM tris pH 7 5 and 2 mM dithiothreitol with reservoir mixture 200 mM CaCl2100 mM Hepes pH 7 5 and 48 PEG 400 Through the entire preparation the channel protein was maintained in solutions containing 150 mM KCl Eor definition of Kquot sites crystals were transferred into solutions where 150 mM KCl was replaced by 150 mM RbCl or 150 mM CsCl 25 M W Tate etaJ Appl CIystaHOgr 28 1961995 D J Thiel eta Rev Sci nstrum 67 i 1996 26 Z Otwinowski in Data COlection and Processing L Sawyer and S Bailey Eds Science and Engineering Research Council Daresbury Laboratory Daresbury UK 1998 pp 56762 27 Collaborative Computational Proiect 4 CCPA Acta CIystaOgr 050 7601994 28 G M Sheldrick ibid 46 467 T990 29 T A Jones J V Zou J Y Cowan M Kieldgaard ibid A47 1100991 80 A T Brunger XVPLOR Version 8 851 Manual The Howard Hughes Medical lnstitute and Department of Molecular Biophysics and Biochemistry Yale Univerr sity New Haven CT 81 S J Gamblin D W Rodgers T Stehle Proceedr ings of the CCP4 study weekend Daresbury Labor ratoryi996 pp 168469 Classical Conditioning and Brain Systems The Role of Awareness Robert E Clark and Larry R Squire Classical conditioning of the eyeblink response perhaps the best studied example of associative learning in vertebrates is relatively automatic and reflexive and with the standard procedure simple delay conditioning it is intact in animals with hippocampal lesions In delay conditioning a tone the conditioned stimulus C8 is presented just before an air puff to the eye the unconditioned stimulus US The US is then presented and thetwo stimuli coterminate ln trace conditioning avariant ofthe standard paradigm a short interval 500 to 1000 ms is interposed between the offset ofthe CS and the onset of the US Animals with hippocampal lesions fail to acquire trace conditioning Amnesic patients with damage to the hippocampal formation and normal volunteers were tested on two versions of delay conditioning and two versions of trace conditioning and then assessed for the extent to which they became aware of the temporal relationship between the CS and the US Amnesic patients acquired delay conditioning at a normal rate but failed to acquire trace conditioning For normal volunteers awareness was unrelated to successful delay conditioning but was a prerequisite for successful trace conditioning Trace conditioning is hippocampus dependent because as in othertasks of declarative memory conscious knowledge must be acquired across the training session Trace conditioning may provide a means for studying awareness in nonhuman animals in the context of current ideas about multiple memory systems and the function of the hippocampus Memory is composed of several different abilities that depend on different brain sys tems A fundamental distinction is be tween the capacity for conscious recollec tion of facts and events declarative or ex plicit memory and various nondeclarative implicit forms of memory that are ex pressed in skills habits and simple forms of conditioning This distinction is dramati cally evident in amnesic patients who have bilateral damage to the hippocampal forma tion or related midline diencephalic brain structures These patients have severely im R E Clark is in the Department of Psychiatry University of California San Diego La Jolla CA 92098 USA L R Squire is at the Veterans Affairs Medical Center San Diego CA 92161 USA and Departments of Psychiatry and Neurosciences University of California San Diego School of Medicine La Jolla CA 92098 USA To whom correspondence should be addressed paired declarative memory and are pro foundly forgetful Yet these same patients have a fully intact capacity for nondeclara tive memory Indeed a large body of literature involving both humans and ex perimental animals can now be understood by recognizing that memory tasks requiring declarative memory depend on the integrity of the hippocampal formation and related structures whereas tasks requiring non declarative memory can be performed nor mally after damage to these structures and are supported by other brain systems De clarative memory is what is meant by the term memory in ordinary language It is involved in modeling the external world and its contents can be brought to con sciousness as a verbal proposition or as a mental image By contrast nondeclarative memory is expressed through performance R 82 K Y J Zhang and P Main Acta CIystaOgr A46 877 88 P J Kraulis J Appl CIystaHOgr 24 946 1991 84 O S Smart J G Neduvelilgtlt Wang B A Wallace M P Sansom J Viol Graphics 14 854 1996 85 We thank D Thiel S Gruner and members of the MacCHESS staff for support and assistance in data collection atAiJ KuriyanS K BurleyS Harrison P Nm E Gouaux and D Wangfor helpful dlSCUSr sions Y Jiangforhelpin data collection D Gadsby and J Kuriyan for comments on the manuscript and T Rahman for patience and support R M is forever grateful to T Wiesel and A L Maclltinnon for making this proiect possible R M is an investigator of the Howard Hughes Medical lnstitute 28 February 1998 accepted 18 March 1998 without affording access to any conscious memory content or even awareness that memory is being used This form of memory permits cumulative changes in perceptual and response systems and allows for the gradual development of new skills and habits A major puzzle about the distinction between conscious hippocampus depen dent and nonconscious hippocampus in dependent forms of memory concerns clas sical conditioning Classical conditioning a phylogenetically early example of simple associative learning has been studied ex tensively and would appear to be a quintes sential example of nondeclarative memory 3 ln perhaps the best studied classical conditioning paradigm delay conditioning of the eyeblink response a neutral condi tioned stimulus CS such as a tone is presented just before an air puff uncondi tioned stimulus US The US is then pre sented and the two stimuli coterminate Fig l A and B Initially an eye blink occurs reflexively in response to the US but with repeated CSUS pairings a learned or conditioned response CR is elicited by the CS in advance of the US The CR overlaps with the US such that the eye blink serves as an adaptive defensive re sponse to the air puff Studies in the rabbit have shown that the cerebellum is essential for both the acquisition and retention of delay classical conditioning 4 and that no other forebrain structure including the hip pocampus is required Amnesic patients also exhibit intact acquisition and retention of the classically conditioned eyeblink re sponse 6 7 Thus eyeblink conditioning appears to have the automatic reflexive features that are characteristic of non declarative memory The puzzle concerns trace conditioning a slightly different version of classical con ditioning in which the CS is presented and terminated and then a short interval is im posed before the presentation of the US 8 Fig l C and D The name comes from the fact that the CS must leave some trace in the nervous system for a CSUS associ wwwsciencemagorg 0 SCIENCE 0 VOL 280 3 APRIL 1998 77
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