heat-inactivated fetal calf serum (FCS). On the nightbefore transfection, cells were plated at 2 3 105 cellsper well. Cells in each well were transfected with 2mg of pMTV-Luc, 2 mg of either MRWT or MRL810, and0.05 mg of pSV2. Transfection was performed withcationic liposomes (Lipofectamine-Plus; Life Technol-ogies) after which cells were incubated in DMEMsupplemented with 10% FCS for 24 hours. Cells werewashed, and then serum-free DMEM containing thetest steroid was added. The cells were incubated foran additional 16 hours before assay. Luciferase andb-Gal activities were measured as described (22).Luciferase activity was normalized to b-Gal activityto correct for transfection efficiency and is expressedas a percentage of the MRWT activity at 1 nM aldo-sterone. All results are the mean of at least nineindependent transfections. For heterodimer experi-ments, the absolute levels of activity induced byaldosterone with MRWT and MRL810 were indistin-guishable, and multiple independent preparations ofboth plasmids yielded indistinguishable results in het-erodimer experiments.

9. R. Rupprecht et al., Eur. J. Pharmacol. 247, 145(1993).

10. D. Moras and H. Gronemeyer, Curr. Opin. Cell Biol.10, 384 (1998).

11. J. F. Dunn, B. C. Nisula, D. Rodbard, J. Clin. Endocrinol.Metab. 53, 58 (1981).

12. Additional data are available at www.sciencemag.org/feature/data/1051091.shl.

13. S. P. Williams and P. B. Sigler, Nature 393, 392(1998).

14. J. Fagart et al., EMBO J. 17, 3317 (1998).

15. The MRWT and MRL810 models were generated fromthe PR crystal structure (13) with the Turbo Frodomolecular graphics program (http://afmb.cnrs-mrs.fr/TURBO_FRODO). Residues abutting the ligand werealtered to match the MR sequence (G773A, M810S,Y941F, and M960L). In addition, the E774G substitu-tion was incorporated to reflect the rotational free-dom G774 confers on helix 3 in MR. The geometrywas regularized with Turbo Frodo. Coordinates foraldosterone (23) were superimposed upon progester-one. Parameters for aldosterone were generated withHIC-Up (24). The 21-hydroxyl group was rotated toform a favorable interaction with the N770 sidechain (14).

16. COS-7 cells were transfected in 100-mm plates with10 mg of receptor plasmid by using Lipofectamine2000 (Life Technologies). On the day after transfec-tion, serum-free media was substituted, and the cellswere grown for an additional 24 hours. Cells wereharvested in 40 mM tris-HCl (pH 7.5), 150 mM NaCl,1 mM EDTA and lysed by freeze-thaw treatment inhypotonic buffer containing 10 mM tris-HCl (pH 7.8),10 mM NaCl, 1 mM EDTA, 10 mM Na2MoO4, 5 mMdithiothreitol, antipain (5 mg/ml), leupeptin (5 mg/ml), chymostatin (5 mg/ml), pepstatin A (5 mg/ml),and 500 mM phenylmethylsulfonyl fluoride. Aftercentrifugation at 15,000g for 15 min, extracts wereadjusted to 100 mM NaCl and 5% glycerol (bindingbuffer). Extracts were incubated overnight with[3H]aldosterone and competitor steroid at 0°C in atotal volume of 200 ml, and then incubated with 100ml of a 50% slurry of hydroxyapatite in bindingbuffer. Samples were centrifuged, washed twice in

binding buffer, and then resuspended in ethanol andprepared for scintillation counting. The value for100% binding was determined by subtracting thenumber of counts per minute bound in the presenceof 500-fold excess of unlabeled aldosterone from thecounts bound in the absence of competitor. Nonspe-cific binding was determined with a 500-fold excessof unlabeled aldosterone. No specific binding wasseen in mock-transfected cells.

17. A. M. Brzozowski et al., Nature 389, 753 (1997).18. W. Bourguet et al., Mol. Cell 5, 289 (2000).19. J. Arriza et al., Science 237, 268 (1987).20. G. M. Lathrop, J. M. Lalouel, C. Julier, J. Ott, Proc. Natl.

Acad. Sci. U.S.A. 81, 3443 (1984).21. R. Rupprecht et al., Mol. Endocrinol. 7, 597 (1993).22. P. Igarashi, D. A. Whyte, K. Li, G. T. Nagami, J. Biol.

Chem. 19, 271 (1996).23. W. L. Duax and H. Haupt, J. Am. Chem. Soc. 94, 5467

(1972).24. G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D54,

1119 (1998).25. We thank members of the family studied for their

invaluable contribution to this project; C. Nelson-Williams, K. Choate, P. Igarashi, R. Denton, and P.Dannies for technical advice; R. Evans for plasmidspRShMRNX and pMTV-Luc; and P. Igarashi for provid-ing pSV2. Supported in part by an NIH SpecializedCenter of Research grant in hypertension. R.P.L. is aninvestigator of the Howard Hughes Medical Institute.Dedicated to the memory of Paul Sigler, whose pas-sion for science remains a source of inspiration.

6 April 2000; accepted 24 May 2000

Structure of the Cytoplasmic bSubunit—T1 Assembly of

Voltage-Dependent K1

ChannelsJacqueline M. Gulbis,*† Ming Zhou,* Sabine Mann,

Roderick MacKinnon‡

The structure of the cytoplasmic assembly of voltage-dependent K1 channelswas solved by x-ray crystallography at 2.1 angstrom resolution. The assemblyincludes the cytoplasmic (T1) domain of the integral membrane a subunittogether with the oxidoreductase b subunit in a fourfold symmetric T14b4

complex. An electrophysiological assay showed that this complex is orientedwith four T1 domains facing the transmembrane pore and four b subunits facingthe cytoplasm. The transmembrane pore communicates with the cytoplasmthrough lateral, negatively charged openings above the T14b4 complex. Theinactivation peptides of voltage-dependent K1 channels reach their site ofaction by entering these openings.

The b subunit of voltage-dependent K1

channels is a tetramer of oxidoreductase pro-teins arranged with fourfold rotational sym-metry like the integral membrane a subunits

(1). Each oxidoreductase protein contains anactive site with catalytic residues and anNADPH (the reduced form of nicotinamideadenine dinucleotide phosphate) cofactor, butthe specific substrate is unknown and thebiological function of the b subunit remains amystery.

Studies of K1 channel biosynthesis haveshown that a and b subunits coassemble inthe endoplasmic reticulum and remain to-gether as a permanent complex (2, 3). Theidea that a large macromolecular assembly isattached to the intracellular face of voltage-dependent K1 channels has important impli-

cations for channel regulation, but it alsoraises the question of how the transmembranepore opens to the cytoplasm. This issue ofpore access first arose when the T1 domain,an about 100–amino acid structure on theintracellular side of the first membrane-span-ning segment of the a subunit, was found toform a tetrameric ring with a narrow, posi-tively charged central pore (4). The small T1pore diameter and positive charge are incon-sistent with functional measurements show-ing that organic cations such as tetraethylam-monium enter the transmembrane pore (5).Even a peptide segment from the channelitself (inactivation peptide) is thought to enterthe pore to produce inactivation (6, 7). Howcan entry of these large molecules be recon-ciled in the setting of a narrow T1 pore? Byanalyzing the structure and function of thecytoplasmic interface, we resolve this appar-ent inconsistency and show how the T1 tet-ramer forms a docking platform for the bsubunit without obstructing the transmem-brane pathway.

It has been postulated that the intracellularT1 domain interacts with the b subunit (8, 9).We reinforced this idea by showing that re-moval of the T1 domain, but not the K1

channel’s COOH-terminus, disrupts b sub-unit association (see below).

Coexpression of the rat b2 subunit (10)(residues 36 to 367) and rat Kv1.1 (11) T1domain (residues 1 to 135) in Sf 9 cells yield-ed a stable complex. The complex was puri-fied and crystallized, and its structure wasdetermined by molecular replacement withthe b subunit structure as a search model(12). The final model was refined to an Rfree

Howard Hughes Medical Institute and Laboratory ofMolecular Neurobiology and Biophysics, The Rock-efeller University, 1230 York Avenue, New York, NY10021, USA.

*These authors contributed equally to this work.†Present address: The Biomolecular Research Insti-tute, 343 Royal Parade, Parkville, Victoria, 3052,Australia.‡To whom correspondence should be addressed. E-mail: mackinn@rockvax.rockefeller.edu

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of 0.229 (40 to 2.1 Å) (12). Packing of sub-units within the crystal defined an unambig-uous interface through which the b subunitand T1 tetramers interact: The large flat sur-face of b engages four prominent loops (con-tact loops) extending from the NH2-terminalside of the T1 tetramer (Fig. 1).

Comparison of the isolated T1 and b sub-unit structural models with their counterpartsin the complex reveals only small differenc-es. One of these is a slight tilt of the T1domain subunits with respect to the centralfour-fold axis (4). The tilt results in a sepa-ration of the subunits at the COOH-terminalside, widening the aperture, and a constric-tion at the NH2-terminal side where T1 con-tacts the b subunit.

To confirm this complex in vivo, we stud-ied the interaction of a and b subunits inXenopus oocytes by measuring inactivation,which refers to occlusion of the pore by aninactivation peptide (Fig. 2A) (6, 10). Theinactivation peptide is effective when at-tached to the NH2-terminus of either the aor b subunits (Fig. 2B), apparently becausethe attachment sites are very near eachother in the T14b4 complex (Fig. 1C).Thus, if the a subunit does not have its owninactivation peptide, trans-inactivation causedby a b subunit indicates a-b subunit asso-ciation. Following a described method (10),a Kv1.4 K1 channel without an inactivationgate (Kv1.4-IR) was coexpressed with theb2 subunit containing an inactivation gate(b12 chimera) (Fig. 2) (13). The T1 do-main of Kv1.4 is 85% identical to that of

Kv1.1 used in the structure determination.When the T1 domain is removed from the

Kv1.4-IR channel by means of genetic dele-tion (13) (residues 2 to 278), inactivation isnot observed in the presence of the b12 chi-mera (Fig. 2C), implying that the T1 domainis necessary for b subunit association. Theoccurrence of inactivation when the inactiva-tion peptide is on the channel’s own NH2-terminus, even in the absence of a T1 domain,demonstrates that the inactivation process it-self does not require a T1 domain [Fig. 2C;see also (14)]. Thus, the T1 domain does notparticipate directly in inactivation but servesto hold the b subunit in place.

To determine which amino acids are im-portant for T14b4 complex formation in vivo,we made single-site alterations (13). On theT1 domain, only mutations involving contactloop residues abolish inactivation (Fig. 2, Dand E). The effect of the point mutations ismost compatible with disruption of b subunitbinding to the a subunit: The same mutationsdo not interfere with inactivation caused by agate on the NH2-terminus of the a subunit(15). On the b subunit, single-site mutationsaffect inactivation only when introduced ontothe surface where the T1 contact loops touchthe b subunit in the crystal structure (Fig. 2,D and E).

Certain mutations on the b subunit [e.g.,Met196 3 Trp (M196W)] reduce the extentof inactivation but do not abolish it (Fig. 2D).In such cases, the inactivating componentexhibits wild-type kinetics, implying thatonly a fraction of channels contain a b sub-

unit because the mutant b subunit has a re-duced affinity for the a subunit. The possi-bility of insufficient mutant b subunit expres-sion, resulting in a mismatch between thenumbers of b and a subunits, is excluded bya control experiment. Mutation M196W onthe b subunit rescues the loss of inactivationcaused by T1 contact loop mutation Phe214

3 Val (F214V) (Fig. 2D). This result offersvery strong support for the crystallographi-cally defined structure in the context of aliving cell. The conclusion that the T1 contactloops provide the b subunit docking surfaceoffers insight into the specificity of a-b sub-unit assembly: The amino acid sequence ofthe T1 contact loop is highly conserved with-in a given family of K1 channels but notbetween members of different families. Thus,b1 and b2 subunits associate with Kv1 butnot Kv2 K1 channels (16).

The aqueous channel down the center ofthe T14b4 complex is too narrow (;4 Å andpositively charged) to allow entry of tetraeth-ylammonium, a pore-blocking cation, or theinactivation peptide (Fig. 3). If not throughthe center of the T14b4 complex, how doesthe ion pathway connect to the cytoplasm?We addressed this question by again exploit-ing inactivation. Inactivation occurs whenan inactivation peptide binds at or near thepore (Fig. 2A). Electrostatic interactionshelp to mediate this process: Mutations ofbasic ( positive charged) amino acids on theinactivation peptide influence the rate andextent of inactivation (17 ). Therefore, weexpect to find complementary acidic (neg-

Fig. 1. Structure of theT14b4 complex. (A)Ribbon representationshowing four contactloops that form theprimary interface be-tween the T1 and btetramers. The T1 tet-ramer is red and the btetramer is blue. (B)Molecular detail of aT1 contact loop touch-ing the b subunit sur-face (24). (C) Stereo-view of a Ca trace ofthe b tetramer (blue)and T1 tetramer (red)viewed along the four-fold axis and showingthe relative proximity of the NH2-termini of each whereinactivation gates are attached. The NADP1 cofactor ineach active site is green. This figure was generated with theprograms O (25), MOLSCRIPT (26), Raster-3D (27), andPOVRAY (28).

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ative charged) amino acids on the channelnear the inactivation peptide-binding site.The T1-S1 linker (connecting T1 to the firstmembrane-spanning segment) contains sev-eral acidic amino acids. When three of theseresidues, located six amino acids COOH-terminal to the T1 structure (Glu273, Asp274,and Glu275 in Kv1.4), are mutated to Ala(neutral) or Lys (positive), the rates of inac-tivation and recovery from inactivation areaffected (18) (Fig. 4, A and B), and mutationsto Lys (negative to positive) have the largestinfluence. Mutation of Val247 to Lys (neutralto positive) also reduced the rate of inactiva-tion (Fig. 4C). Val247 is located on the sur-face of the T1 domain close to where the

T1-S1 linker emerges (Fig. 5).Mutations were also made in the inactiva-

tion peptide to reduce its positive charge.These mutations affected inactivation, butmore importantly their influence depended onthe charge at positions 273 to 275 in theT1-S1 linker. Thus, mutations in the inacti-vation peptide are coupled in mutant cycleanalysis to mutations in the T1-S1 linker nearthe T1 domain (Fig. 4C) (19). In order forsuch coupling to occur, the peptide must benear the T1-S1 linker when it inactivates thepore. Because the peptide cannot fit throughthe center of the T14b4 complex, it mustreach the pore by entering lateral openingsabove the T1 tetramer (Fig. 5).

The structure of the T1 domain tetramerseemed to be inconsistent with several de-cades of K1 channel pharmacology: The cen-tral hole is too narrow for many of the clas-sical organic blocking agents (4). When theT1 domain was deleted from the a subunit,the single channel conductance did not changesubstantially, as if the T1 tetramer does notform an extension of the pore (20). Thus, itwas proposed that either the T1 domain is nota tetramer in the context of the integral mem-brane channel or that ions move between thecytoplasm and pore through an alternativepathway.

The data presented here establish that theT1 domain is indeed a tetramer in the K1

Fig. 2 (left). Functional analysis of the T14b4 interface. (A) Illustration ofN-type K1 channel inactivation. The black circle represents the inacti-vation peptide. (B) Potassium currents recorded from Xenopus oocytesunder voltage clamp containing channels without an inactivation peptide(Kv1.4-IR) or with an inactivation peptide attached to the NH2-terminusof the a subunit (Kv1.4-b1N) or b subunit (Kv1.4-IR 1 b12). The a andb subunits were coexpressed at an RNA ratio of 1:2 (v:v) (13). (C) TheKv1.4D278 channel lacks amino acids 2 to 278, which make up Kv1.4channel’s own inactivation peptide and T1 domain. The b12 chimera failsto cause inactivation (Kv1.4D278 1 b12). The Kv1.4D31-286 channel(amino acids 31 to 286 deleted) lacks a T1 domain but contains its owninactivation peptide (Kv1.4D31-286), which differs from that of b12,accounting for the slower inactivation rate. (D) (Top) A point mutationon the contact loop of the T1 domain (Kv1.4-IR F214V) prevents b12chimera–induced inactivation (residue Phe73 in Kv1.1) (Middle) Currentscarried by Kv1.4-IR channels expressed with a mutant b subunit (b12M196W) at two RNA volume ratios, 1:10 and 1:50, undergo incompleteinactivation. (Bottom) Coexpression of Kv1.4-IR F214V mutant a subunitand b12 M196W mutant b subunit yields inactivation properties similarto the combination of wild-type a and b subunits. In all recordings,oocytes were held at 280 mV and stepped to 160 mV for 200 ms. (E)GRASP view of T1 and b2 subunits showing the effect of selected pointmutations on channel inactivation (red, alteration; blue, no alteration).(Top) NH2-terminal surface of the T1 domain. Red residues are Phe73,Leu76, and Arg78, and the blue residue is Pro75. (Middle) Flat surface ofthe Kvb2 subunit. Red residues are Met196, Tyr199, Ser200, and Gln204, andblue residues are Arg203, Lys268, Lys129, and Met193. (Bottom) Concave

surface of the b subunit. Blue residues are Asp66, Asp67, Tyr90, Lys104, Lys105, Lys106, Lys124, Glu145, Gln148, Glu150, Asn158, Arg159, Arg189, Pro260, andPro261. This figure was made with GRASP (29). Fig. 3 (right). The central pore of the T14b4 complex. A molecular surface calculated over threeof the four T1-b subunits reveals a central pore with unusual internal electrostatic features. The pore is coincident with the molecular four-fold axisand extends the length of the complex. At its apex, it is sufficiently wide to permit the entry of ions. The inner surface of this cavity is lined withcharged amino acid side chains that are conserved in the Kv1 family. Calculation of the electrostatic potential was carried out at an ionic strengthequivalent to 0.15 M KCl. Dielectric constants of 2.0 for the protein interior and 80.0 for solvent were used. Regions of the molecular surface exhibitingintense negative charge are colored red, and electropositive regions are colored blue. This figure was generated with the program GRASP (29).

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channel and that the T1 domain tetramerforms the docking platform for the b subunit.The contact loops extending from the NH2-terminal face of the T1 domain are obviousstructures for interfacing with intracellularproteins—in this case the b subunit. We sug-gest that the major role of the T1 domain is toform a docking site for proteins whose activ-ities are coupled to K1 channel function.

The apparent paradox of a too narrowpore in the T1 domain has a simple explana-tion. Our data suggest the presence of lateralopenings between the T1 domain and theintegral membrane part of the a subunit (Fig.5). This explanation accounts for the interac-tion between the inactivation peptide andamino acids on the top (COOH-terminal face)of T1 and in the T1-S1 linker close to T1.Because the peptide must access the porethrough lateral openings, K1 ions must alsopass through the openings. Lateral openings inthe cytoplasm, with negative charged aminoacids, are also a structural feature of nicotinicacetylcholine receptor ion channels (21).

A single inactivation gate binds to its recep-tor near the intracellular pore entryway in anexclusive fashion when inactivation occurs (22,

Fig. 4. Evidence for pore openings above the T1domain. (A) (Top) b12 chimera–induced inactiva-tion of wild-type Kv1.4-IR a subunits (WT ) and asubunits with Ala (A) or Lys (K) substituted atpositions 273 to 275. (Bottom) Currents fromwild-type a subunits (WT) or a subunits with Alasubstituted at 273 to 275 (A) coexpressed withthe b12 chimera containing a wild-type inactiva-tion gate (1b12) or K13N, R15G mutant inacti-vation gate (11315NG). The experimental condi-tions were identical to those described in Fig. 2D.(B) (Top) Rate of recovery from inactivation mea-sured in paired pulses from a holding voltage of280 mV and stepping the membrane to 160 mV.The initial conditioning pulse was 400 ms. Thedashed curve corresponds to an exponential func-tion (t 5 11 s) fitted to the envelope defined bythe peak current of the test pulse. (Bottom) Frac-tion of recovery as a function of time between theconditioning pulse and the test pulse. f, Kv1.4-IRa subunit paired with the b12 chimera; v, asubunit with Ala substituted at 273 to 275 coex-pressed with b12 chimera; V, a subunit with Lyssubstituted at 273 to 275 coexpressed with b12chimera. The solid lines are single exponential fits.Error bars are the standard error of the mean(SEM) from $five independent experiments. (C)Double-mutant cycle analysis of inactivation. Thetime constants of inactivation (tinac) and recovery(trec) and the ratio between these two time con-stants are tabulated (6SEM, $five independentexperiments) (18). The V value was calculated asdescribed (19).

Fig. 5. Composite mod-el of a voltage-depen-dent K1 channel. The asubunit is shown in red,and the b subunit is inblue. The model of thepore region is based onthe KcsA K1 channel(30). The structures ofthe voltage-sensing re-gion and connectorsare unknown (depictedschematically). An NH2-terminal inactivationpeptide is shown enter-ing a lateral opening togain access to the pore.Proposed locations ofamino acids 273 to 275on the T1-S1 linker areshown (black asterisks)with the location ofVal247 on the T1 do-main surface (greenasterisks).

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23). Our findings are entirely consistent withthis conclusion. Although we expect there to befour openings above the T1 domain, there ispresumably only one site near the pore entry-way where a single peptide can induce inacti-vation. We propose that the inactivation peptideis poorly structured and snakes its way into alateral opening to reach the central pore and thisis why mutations in the T1-S1 linker influencethe process. In some cases, four peptides mayeven be bound above T1, poised and ready,before inactivation.

The central question as to why K1 chan-nels contain an oxido-reductase enzyme sub-unit remains unanswered. In this report, wehave shown how the b subunit attaches to avoltage-dependent K1 channel through itsinteraction with the T1 domain. We previous-ly suggested that interactions between a andb subunits may allow cellular redox regula-tion of the channel or channel regulation ofenzyme activity (1). The structure of theT14b4 complex takes us one step closer tounderstanding how such energetic couplingmight occur.

References and Notes1. J. M. Gulbis, S. Mann, R. MacKinnon, Cell 97, 943

(1999).2. G. Shi et al., Neuron 16, 843 (1996).3. N. Nagaya and D. M. Papazian, J. Biol. Chem. 272,

3022 (1997).4. A. Kreusch, P. J. Pfaffinger, C. F. Stevens, S. Choe,

Nature 392, 945 (1998).5. C. M. Armstrong, J. Gen. Physiol. 58, 413 (1971).6. W. N. Zagotta, T. Hoshi, R. W. Aldrich, Science 250,

568 (1990).7. S. D. Demo and G. Yellen, Neuron 7, 743 (1991).8. S. Sewing, J. Roeper, O. Pongs, Neuron 16, 455

(1996).9. W. Yu, J. Xu, M. Li, Neuron 16, 441 (1996).

10. J. Rettig et al., Nature 369, 289 (1994).11. A. Baumann, A. Grupe, A. Ackermann, O. Pongs,

EMBO J. 7, 2457 (1988).12. DNA encoding the NH2-terminal cytoplasmic T1 do-

main (residues 1 to 135) of the rat Kv1.1 a subunitwas subcloned into a pAcG2T (Pharmingen) baculo-virus transfer vector as a glutathione S-transferase(GST ) fusion construct with Bam HI and Eco RIrestriction sites. DNA encoding residues 36 to 367 ofrat Kvb2 was subcloned into pFastBac1 (Gibco) be-tween Bam HI and Eco RI restriction sites. For coex-pression, cells were infected with two viruses. At 72hours, cells were harvested by centrifugation andresuspended in a cold buffer (I) comprising 20 mMtris-Cl (pH 8.0), 150 mM KCl, 5 mM MgCl2, and 5 mMdithiothreitol. Phenylmethylsulfonyl fluoride wasadded to 1 mM, and the cells were promptly lysedwith a French pressure cell. After adding deoxyribo-nuclease, the lysate was incubated for 20 min on iceand then centrifuged to pellet cellular debris. Thefiltered lysate was incubated with glutathione sepha-rose 4B beads (Pharmacia) for 2 hours at 4°C. Pro-teolytic (thrombin) removal of the GST tag waspreceded by extensive washing of the matrix-boundmaterial with a buffer (II) containing 20 mM tris-Cl(pH 8.0), 150 mM KCl, and 2 mM CaCl2. The cleavedprotein was eluted, concentrated, and loaded onto asize-exclusion (Superdex-200 10/30; Pharmacia) col-umn equilibrated in buffer (I). Peak fractions corre-sponding to a heterooctamer ( T14b4) were pooledand cleaved with trypsin, yielding a T1(27-129)4b4octamer. Size exclusion was used as a final purifica-tion step. Column fractions corresponding to theheterooctameric complex were collected, pooled, andconcentrated to 15 mg/ml. Tetragonal crystals con-taining a single T1b heterodimer (P4212; a 5 100.7

Å, c 5 110.9 Å) grew in hanging drops; 1 ml of theprotein solution was mixed with 1 ml of a reservoirsolution containing 50 mM glycine (pH 8.6) and 6%PEG400 and allowed to equilibrate by vapor diffu-sion. Crystals were cryoprotected by transfer into asimilar solution containing 25% glycerol, mounted innylon loops, and frozen in propane. Diffraction datato 2.1 Å were collected from single plates at beam-lines A1 (CHESS) and X25 (Brookhaven NSLS) withcharge-coupled device detectors. Images were pro-cessed with DENZO and intensities were mergedwith SCALEPACK [Z. Otwinowski, in Proceedings ofthe CCP4 Study Weekend (SERC Daresbury Laborato-ry, Warrington, UK, 1993), pp. 56–62; Z. Otwinowskiand W. Minor, Methods Enzymol. 276, 307 (1997)]. Aset of 400,177 measured reflections (33,907 unique)in the resolution range 40.0 to 2.1 Å gave a 98.8%complete data set (92.9% in the outer shell; 2.15 to2.10 Å). The overall agreement factor Rmerge (onintensity) is 8.0% (30.0% in the outer shell). Theaverage intensity/standard deviation is 17.8 (4.3 inthe outer shell). The T14b4 structure was solved bymolecular replacement with AMORE [ J. Navaza, ActaCrystallogr. Sect. A 50, 157 (1994)]. A refined modelof the conserved core of rat Kvb2 (1qrq; residues 36to360) was used to initiate rotation and translationsearches in the resolution range 8.0 to 4.0 Å. A peak8.8s above the mean was the highest in the rotationfunction. Translation searches yielded a peak at18.0s. Cycles of rigid body refinement of the het-erodimer with data between 8.0 and 4.0 Å reducedthe R factor to 0.393 and gave a correlation coeffi-cient of 57.7. Before refinement, all temperaturefactors were fixed at 30 Å2, and several residues inmobile regions together with several side chains weredeleted from the model. The b subunit T1 complexmodel was then refined by minimization and simu-lated annealing procedures with the maximum like-lihood target implemented in CNS [A. T. Brunger etal., Crystallogr. D. Biol. Crystallogr. 54 (part 5), 905(1998)]. 3Fo 2 2Fc (where Fo and Fc are the observedand calculated structure factors, respectively) mapswere calculated on the basis of an average of 10refined structures and were of sufficiently high qual-ity to enable almost complete tracing of the T1domain. A number of cycles of building and refine-ment were required to place the remainder of thestructure. All data falling between resolution limits of40.0 and 2.1 Å, with the exception of a random 5%used for calculation of the Rfree, were used in therefinement. Bulk solvent and anisotropic temperaturefactor corrections (final anisotropic tensor elements:B11, 0.26 Å2; B22, 0.26 Å2; and B33, 20.52 Å2) wereapplied to the reflection data. The crystallographicRfree was monitored to reduce model bias. Individualtemperature factors were refined for all nonhydrogenatoms. The refined model has good geometry withone Ramachandran outlier near NADP and contains418 residues and 1 nicotinamide adenine dinucleoti-de phosphate (NADP1) and 267 water molecules andhas an Rfree of 0.229 and an Rw of 0.211. Six residuesat the COOH-terminus of the b subunit, eight at theNH2-terminus of T1, and three at the COOH-termi-nus of T1 were omitted from the model because ofweak electron density in the region. Side chains werenot modeled beyond the b carbon for the followingamino acids: Lys124, Lys268, and Ser361.

13. The Kv1.4-IR (residues 2 to 110 deleted), Kv1.4D278(residues 2 to 278 deleted), and Kv1.4D31-286 (res-idues 31 to 286 deleted) were generated by theoverlapping polymerase chain reaction method. Theb12 chimera and the Kv1.4-b1N were made by splic-ing DNA encoding residues 1 to 70 of b1 onto DNAencoding residues 36 to 366 of b2 or DNA encodingresidues 137 to 655 of Kv1.4, respectively. All con-tructs were cloned into pBluescript KS1. Point muta-tions were made by the QuickChange method (Strat-agene) and confirmed by sequencing the entire in-sertion. RNA was prepared by T7 polymerase tran-scription and injected into Xenopus laevis oocytes [Z.Lu and R. MacKinnon, J. Gen. Physiol. 104, 477(1994)]. RNA ratios refer to volumes mixed beforeinjection. K1 currents were recorded under two-electrode voltage clamp (OC725B; Warner Instru-ment, Hamden, CT ). Electrodes were drawn from

borosilicate glass capillaries (World Precision Instru-ment, Sarasota, FL) to a resistance of ;0.5 megaohms(3 M KCl). Bath solution contained 96 mM NaCl, 2mM KCl, 0.3 mM CaCl2, 1 mM MgCl2, and 5 mMHepes (pH 7.6) with NaOH. Analog data from theamplifier were filtered (2 kHz, 23 dB) with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA),digitized at 10 kHz, and transferred to a computerhard disk with an Axon 1200 digitization board andsupporting software (Clampex version 8; Axon Instru-ment, Foster City, CA).

14. T. E. Lee, L. H. Philipson, D. J. Nelson, J. Membr. Biol.151, 225 (1996).

15. J. M. Gulbis, M. Zhou, S. Mann, R. MacKinnon, datanot shown.

16. K. Nakahira, G. Shi, K. J. Rhodes, J. S. Trimmer, J. Biol.Chem. 271, 7084 (1996).

17. R. D. Murrell-Langnado and R. W. Aldrich, J. Gen.Physiol. 102, 949 (1993).

18. Inactivation time constants were obtained by fitting thecurrent decay with exponential functions with Clampfit(Axon) software. Expression of Kv1.4-IR with b12 chi-mera yielded two exponentials: a predominant (.90%)fast component (time t , 60 ms) and a minor (,10%)component (t ; 500 ms). The slower time constantwas relatively invariable and coincided with the COOH-type inactivation of Kv1.4-IR [S. H. Heinemann, J. Rettig,H. R. Graack, O. Pongs, J. Physiol. 493, 625 (1996)]. Indouble-mutant cycle analysis, only the predominantcomponent was used. Inactivation recovery was studiedwith paired pulses (22). The peak of the second pulse inthe pair was graphed as a function of time after the firstpulse, defining an envelope of recovery that was fit toan exponential function with Origin (Microcol Software,Northampton, MA). Recovery from inactivation forKv1.4-IR paired with the b12 chimera was well de-scribed by single exponential function.

19. The double-mutant cycle parameter

V 5Kd

wt,wt 3 Kdmut,mut

Kdwt,mut 3 Kd

mut,wt

was used to quantify the degree of coupling betweentwo mutations [P. Hidalgo and R. MacKinnon, Science268, 307 (1995)]. Here, Kd refers to a dimensionlessparameter defined by the ratio of inactivation andrecovery time constants, tinac/trec. Because trec ..tinac, Kd is very close to the equilibrium ratio of opento inactivated channels. A value of V greater thanunity indicates that the effects of two mutations arecoupled.

20. W. R. Kobertz and C. Miller, Nature Struct. Biol. 6,1122 (1999).

21. A. Miyazawa, Y. Fujiyoshi, M. Stowell, N. Unwin, J.Mol. Biol. 288, 765 (1999).

22. R. MacKinnon, R. W. Aldrich, A. W. Lee, Science 262,757 (1993).

23. F. Gomez-Lagunas and C. M. Armstrong, Biophys. J.68, 89 (1995).

24. Single-letter abbreviations for the amino acid resi-dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F,Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn;P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; andY, Tyr.

25. T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ActaCrystallogr. Sect. A 47, 110 (1991).

26. P. Kraulis, J. Appl. Crystallogr. 24, 946 (1991).27. D. Bacon and W. F. Anderson, J. Mol. Graph 6, 219

(1988).28. See www.povray.org.29. A. Nicholls, K. A. Sharp, B. Honig, Proteins 11, 281

(1991).30. D. A. Doyle et al., Science 280, 69 (1998).31. We thank J. Bonnano for technical assistance; J. Morais

Cabral, R. Dutzler, Y. Jiang, A. Pico, and Y. Zhou for helpwith data collection; B. Chait and M. Cadene for massspectrometry; O. Pongs for Kv1.4 DNA; J. Trimmer for bsubunit DNA; members of the MacCHESS (A1) andBrookhaven National Laboratory (X25) staff for assist-ance in data collection; and W. Chin for artwork andhelp with the manuscript. The coordinates have beensubmitted under Protein Data Bank ID code 1EXB. R.M.is an investigator in the Howard Hughes Medical Insti-tute. Supported by NIH grant GM47400.

21 April 2000; accepted 1 June 2000

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