Locations of the �1 transmembrane helicesin the BK potassium channelGuoxia Liu*†, Sergey I. Zakharov*†, Lin Yang*, Roland S. Wu*, Shi-Xian Deng‡, Donald W. Landry‡, Arthur Karlin§¶,and Steven O. Marx*¶�

Divisions of *Cardiology and ‡Experimental Therapeutics, Department of Medicine, §Center for Molecular Recognition, Departments of Biochemistry,Physiology, and Neurology, and �Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032

Contributed by Arthur Karlin, May 30, 2008 (sent for review April 9, 2008)

BK channels are composed of �-subunits, which form a voltage-and Ca2�-gated potassium channel, and of modulatory �-subunits.The �1-subunit is expressed in smooth muscle, where it renders theBK channel sensitive to [Ca2�]i in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BKchannel acts thereby as a damped feedback regulator of voltage-dependent Ca2� channels and of smooth muscle tone. We exploredthe contacts between � and �1 by determining the extent ofendogenous disulfide bond formation between cysteines substi-tuted just extracellular to the two �1 transmembrane (TM) helices,TM1 and TM2, and to the seven � TM helices, consisting of S1–S6,conserved in all voltage-dependent potassium channels, and theunique S0 helix, which we previously concluded was partly sur-rounded by S1–S4. We now find that the extracellular ends of �1TM2 and � S0 are in contact and that �1 TM1 is close to both S1 andS2. The extracellular ends of TM1 and TM2 are not close to S3–S6.In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2shifted the conductance–voltage curves toward more positivepotentials, slowed activation, and speeded deactivation, and ingeneral favored the closed state. TM1 and TM2 are in position tocontribute, in concert with the extracellular loop and the intracel-lular N- and C-terminal tails of �1, to the modulation of BK channelfunction.

auxiliary subunit � cysteine substitution � disulfide cross-linking �electrophysiology � mslo1

BK potassium channels have large conductances and areuniquely both voltage- and Ca2�-activated. They are nega-

tive feedback regulators of cellular excitability and of [Ca2�]i. BKchannels are a complex of four �-subunits (1) and four �-sub-units (2–7). In addition to the S1–S6 transmembrane (TM)helices conserved in all voltage-gated K� channels, � contains aunique seventh TM helix, S0, N-terminal to S1–S6; furthermore,the first 19 residues preceding S0 are extracellular (Fig. 1A) (8).A large cytoplasmic domain, C-terminal to S6, contains twoRCK domains, resembling those of the bacterial Ca2�-gated K�

channel, MthK (9). The voltage-sensitivity of the BK channel isa property of the membrane domain of �, whereas the Ca2�-sensitivity is conferred by the C-terminal cytoplasmic domain (10).

The channel formed by BK � is modulated by �-subunits.There are four types of �-subunits, �1, �2, �3, and �4, 191–235residues in length (2–6). �-Subunits have short cytoplasmic N-and C-terminal segments, two TM helices, TM1 and TM2, anda large extracellular loop between them (Fig. 1B). The �1-subunit is expressed in smooth muscle. In the presence of Ca2�,it shifts the V50 for channel activation toward the resting poten-tial, around which channel opening becomes a roughly linearfunction of [Ca2�]i in the physiologically relevant 1–10 �Mrange. In addition, �1 slows both activation and deactivation.Activation of BK channels shifts the membrane potential tomore negative values, suppressing the activity of L-type Ca2�

channels, thereby suppressing Ca2� influx. Thus, the BK channelacts as a regulator of [Ca2�]i and, consequently, of smoothmuscle tone.

The other � types have some similar and some different effectson channel function. Like �1, �2 and �4 slow gating kinetics and,in the presence of Ca2�

, shift the G–V curve in the hyperpolar-izing direction (2). The �2-subunit (6) and some variants of �3(11) also confer N-type inactivation. The �3-subunit also confersrectification (11, 12).

In addition to the rectification by �3, the interaction of eachof the �s with charybdotoxin and iberiotoxin binding (13) and itseffects on � (14), as well as the cross-linking of charybdotoxin to�1 (15), are consistent with some part of the extracellular loopsof � overlapping the ion permeation pathway. Evidence for arole of the extracellular loop in gating is that a point mutationin the human �1 extracellular loop shifts the V50 to a morenegative potential (16).

A role for the membrane domain of � is suggested by thedependence of �1 modulation of the G–V curve on the speciesof � S0, as tested in Drosophila–human chimeras of �; it wassuggested that S0 and its preceding extracellular N-terminalresidues act as a docking site for �1 (8). Deletion of the 20extracellular, N-terminal amino acid residues of � disrupted the�1-induced, but not �2-induced, hyperpolarizing shift in the G–Vrelationship (17). This deletion did not affect �1 slowing ofgating kinetics.

The intracellular N- and C-terminal tails of � play major rolesin their modulation of �, beyond inactivation mentioned above.

Author contributions: G.L., S.I.Z., A.K., and S.O.M. designed research; G.L., S.I.Z., L.Y., andR.S.W. performed research; S.-X.D. and D.W.L. contributed new reagents/analytic tools;G.L., S.I.Z., L.Y., R.S.W., A.K., and S.O.M. analyzed data; and A.K. and S.O.M. wrote thepaper.

The authors declare no conflict of interest.

†G.L. and S.I.Z. contributed equally to this work.

¶To whom correspondence may be addressed. E-mail: ak12@columbia.edu or sm460@columbia.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0805212105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

Fig. 1. Mouse BK �- and �1-subunits. (A) Scheme of the threading of BK �

through the membrane. The extracellular regions flanking S0–S6, in which Cyswere substituted, are indicated by thick lines. (B) Scheme of the threading ofBK �1 through the membrane. The extracellular regions flanking TM1 andTM2, in which Cys were substituted, are indicated by thick lines. Two disulfidebonds within the extracellular loop are shown.

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Deletion in �1 of either tail separately or of both tails eliminatedthe leftward shift in the G–V curve (18). Using chimeric �1- and�2-subunit constructs, Latorre and colleagues (19) found thatdifferences in the shift and slope of the G–V curves anddeactivation properties of these two � types depend mainly onthe N- and C-terminal tails. Consistent with an important rolefor the � intracellular domains, substitution of a portion of theBK � C-terminal, intracellular domain with the related portionof the Slo3 potassium channel prevented �1 modulation ofchannel function (20).

To obtain more direct information on the location of � relativeto �, we determined the positions of the extracellular ends of �1TM1 and TM2 relative to the extracellular ends of � S0–S6. Ourapproach was similar to the one we used to determine thelocation of the extracellular end of S0 relative to S1–S6 (21). Wedetermined the extent of endogenous disulfide bond formationbetween cysteines (Cys) substituted, one at a time, for each of thefirst four (or more) residues just f lanking the extracellular endsof TM1 and TM2 in �1 and for each of the four (or more)residues just f lanking the extracellular ends of S0–S6 in �. EachCys-substituted mutant of � and each Cys-substituted mutant of�1 were coexpressed in HEK293 cells. The cell-surface BKchannels were analyzed for the extent of endogenous disulfidebond cross-linking of � and �1. The functional consequences ofthe mutations to Cys and of the disulfide between the mutant �and mutant �1 were determined in representative pairs. Wefound that the extracellular end of �1 TM1 is close to both S1and S2, and the extracellular end of �1 TM2 is close to S0.

ResultsMutant �- and �1-Subunits. We mutated to Cys, one at a time, thefirst four (or more) residues just extracellular to the membranedomain of S0–S6 in pseudo-wild-type (pWT) � [Fig. 1 A andsupporting information (SI) Fig. S1 A] and TM1 and TM2 inpWT �1 (Fig. 1B and Fig. S1B). In the polar juxtamembraneenvironment, the Cys thiol can ionize to the reactive thiolate andis accessible to oxidizing enzymes and reagents. Furthermore,Cys just outside the membrane domain of different TM helicesare more or less in the same plane parallel to the membrane (Fig.1). Because at least four consecutive positions are mutated, alldirections parallel to the membrane are sampled, whatever thelocal secondary structure, and in neighboring segments at leastsome substituted Cys are likely to be pointing toward each otherand susceptible to disulfide bond formation.

The TM helices in � were predicted originally by Wallner et al.(8), and those in BK �1 were predicted by Knaus et al. (3). Inboth � and �1, the PHDhtm algorithm (22) predicted similarboundaries between membranous and extramembranous do-mains of the TM helices. Although the predicted BK � S1–S6helices align with the homologous S1–S6 of KvAP (23) and Kv1.2(24), the boundaries of the membrane-embedded domains arenot precisely defined in these channels crystallized from deter-gent solution.

BK � contains two extracellular Cys, Cys-14 and Cys-141,which we previously showed form a disulfide bond in the nativestructure (21). We mutated each of these Cys to Ala to generateour background construct, pWT �. Whether alone or combinedwith �1, pWT � and wild-type (WT) � function the same (21).

BK �1 contains two Cys in TM1 (Fig. S1B), which we mutatedto Ala to generate our background construct, pWT �1. The fourCys residues in the extracellular loop of �1, conserved in all �types, which were hypothesized to form disulfide bonds (25),were not mutated. WT �1 and pWT �1 have indistinguishableeffects on the function of WT � and pWT � (see below).

Most combinations of 30 Cys-substitution mutants of pWT �and 10 Cys-substitution mutants of pWT �1 were coexpressed inHEK293 cells. Two days after transfection, the cells weresurface-labeled with a relatively impermeant biotinylation re-

agent, and the biotinylated cell-surface proteins were solubilized,captured on avidin beads, and released in nonreducing samplebuffer for SDS/PAGE. Judging from the densities of immuno-detected bands, we concluded that all � mutants, except forA295C and T297C, and all �1 mutants, except for K44C, wereexpressed on the cell surface in amounts similar to pWT � andpWT �1, respectively. The three poorly expressing mutants werenot analyzed further.

As in our previous study of the location of � S0 relative toS1–S6 (21), we added no cross-linking reagents; rather, wedetermined the extent of endogenous disulfide bond formation.

Disulfide Cross-Linking of Cys-Substituted Mutants of � and �1.Heterologously expressed �-subunit has an apparent molecularmass of �125 kDa. Immunoblots of �1-subunit show two orthree bands, the principal one of apparent molecular mass 25kDa. The heterogeneity in �1 is likely due to variable glycosyl-ation. The complex of cross-linked Cys-substituted � and Cys-substituted �1 runs as a broad band with an apparent molecularmass of �150 kDa (Fig. 2). No band at 150 kDa resulted aftercoexpressing pWT � and pWT �1 (Fig. 2), or any of theCys-substituted �s and pWT �1, or any Cys-substituted �1s andpWT � (data not shown).

The band at 150 kDa contains � cross-linked to �1: This banddisappears after reduction of the sample with DTT, and themonomeric 125-kDa � band appears (Fig. 2). In addition,coexpressed � and �1 Cys mutants were extracted and eitherreduced with DTT or not. Both samples were immunoprecipi-tated with an anti-�1 antibody; the complex was dissociated andfully reduced in SDS, electrophoresed, and immunoblotted withanti-� antibody (Fig. S2). The 125-kDa � band was immuno-precipitated with anti-�1 antibody only from the initially unre-duced sample and not from the initially reduced sample.

The endogenously cross-linked Cys mutants of � and �1expressed on the cell surface could be reduced and reoxidized onthe cell surface with a membrane-impermeant, quaternary am-monium diamide derivative, QPD (21). This is demonstratedwith three pairs of Cys-substituted � and �1 (Fig. S3).

Patterns of Disulfide Bond Formation. The extents of disulfide bondformation between the different f lanking segments variedwidely. Cys substituted in the TM1 flank formed disulfides mostreadily with Cys in the flanks of S1 and S2 (Fig. 3A), whereas Cysin the flank of TM2 formed disulfides most readily with Cys inthe flank of S0 (Fig. 3B). There was no significant disulfide bondformation between the flanks of TM1 or TM2 and the flanks ofS3, S4, S5, or S6. In all Cys pairs involving the flanks of S3–S6,except one, the extent of cross-linking was �10%. The exceptionwas L41C in the TM1 flank and G260C, which were 20%cross-linked; G260, however, is eight residues from the predicted

Fig. 2. Detecting cross-linking between BK �- and �1-subunits. Western blotsillustrating endogenous cross-linking of pairs of control and Cys-substituted �-and �1-subunits. Constructs were expressed in HEK cells, and proteins wereextracted, denatured in SDS, and either reduced with DTT or not. The blotswere developed with anti-BK � antibody reacting with both � (125 kDa) andthe cross-linked �–�1 dimer (�150 kDa). At the bottom of each gel is thefraction of cross-linked �–�1 dimer before reduction.

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emergence of S5 from the membrane (Fig. 3A; note that theresidues closest to the membrane are underlined). Cys substi-tuted in the S0 flank cross-linked to Cys substituted in the TM2flank to a high extent in all of the combinations, and the Cys inpositions closest to the membrane cross-linked to a slightlygreater extent than those further out (Fig. 3B). In the othercombinations, Cys substituted for the residues three or fourpositions out from the membrane tended to form disulfides to agreater extent than Cys substituted for residues one or twopositions out from the membrane.

We take the extent of disulfide bond formation between twoCys as a measure of their relative proximity (see Discussion). Toestimate the relative proximity of a pair of flanks, we averaged

the top three extents of cross-linking of Cys in the two flanks.These averages (� SD) were, for TM1 to S0, 42 � 8%, for TM1to S1, 71 � 7%, and for TM1 to S2, 74 � 13%. The averages were,for TM2 to S0, 86 � 3%, for TM2 to S1, 16%�1%, and for TM2to S2, 36 � 4%. In a model of BK �, we have placed theextracellular ends of TM1 and TM2 among the extracellular endsof S0–S6 in positions consistent with these average extents ofcross-linking and with the near absence of cross-linking of TM1and TM2 to S3–S6 (Fig. 4).

Functional Effects of Cross-Links. We examined the function ofhighly cross-linked �- and �1-subunits to determine whether thestructure of the cross-linked complex was near-native and that,therefore, the extent of cross-linking reflected proximity in anear-native structure. Our assumption was that near-nativefunction implies near-native structure. All tested pairs of Cys-substituted � and Cys-substituted �1 were functional, albeitsomewhat perturbed. A second question was whether theseperturbations provided clues about functional mechanisms. Thebaseline for normal function was that of the channel formed bypWT � and pWT �1, which was identical to WT � and WT �1in the functional properties that we tested; namely, the G–Vcurve at 10 �M Ca2�� and the rate constants for activation(opening) and deactivation (closing) (Fig. S4A) (21).

We characterized the function of highly cross-linked mutant �and mutant �1 pairs both before and after reduction with DTT.Both before and after reduction, the population of channelsconsisted of a mixture of disulfide cross-linked subunits andun-cross-linked subunits. Our aim was to quantitate the effect onfunction of the replacement of two native residues by cysteinescross-linked by a disulfide (a cystine) and separately the effect onfunction of the replacement of these two native residues by

Fig. 3. Mean extents of endogenous disulfide-bond formation between Cyssubstituted in the extracellular flanks of � and �1. (A) � S0–S6 flanks and �1TM1 flank. (B) � S0–S6 flanks and �1 TM2 flank. The � residues substituted byCys are indicated along the left edge of each vertical axis, and the �1 residuessubstituted by Cys are color-coded as shown. The extent of disulfide-bondformation (%) is represented by bars in the horizontal direction. In the casesin which the mean extent of disulfide-bond formation was zero, the value0.5% was plotted to identify these pairs as tested. The residues closest to themembrane are underlined.

Fig. 4. Proposed location of the extracellular ends of �1 TM1 and TM2relative to the extracellular ends of � S0–S6. We have taken a model of thestructure of Kv1.2 in the closed state (30) as a template for the structure of BK� S1–S6. The white-filled circles represent the extracellular ends of �1 TM1 andTM2 and are placed according to the means of the top three extents ofcross-linking to the � TM helices. The position of � S0 relative to S1–S6 wasdetermined as described in ref. 21. The circles are connected by color-codedlines, shown only in the blue �-subunit, representing the binned averageextents of endogenous cross-linking: light blue, 5–19%; green, 20–39%;yellow, 40–59%; orange, 60–79%; red, 80–100%.

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reduced cysteines. We determined the effects on three param-eters, V50; the rate constant for channel activation, kACT; and therate constant for channel deactivation, kDEACT. The effect of V50,designated �V50, was taken as V50,MUTANT � V50,pWT. The effectson the rate constants were taken as log(kMUTANT/kpWT). Toestimate the effects separately of the disulfide and of the doublemutation, we made a simplifying assumption. We assumed thatthe observed effect was a weighted sum of the effect due to thereplacement of two native residues by a cystine and the effect dueto the replacement of two native residues by reduced cysteines;the weights were the proportions of the disulfide cross-linkedform and of the reduced form as determined by Western blotanalysis (Fig. 2). We assume this linearity of contributions to theoverall effect even though individual channels will contain amixture of cross-linked and un-cross-linked subunits. This al-lowed us to write two simultaneous equations for the observedeffects before DTT and after DTT and to solve for the pureeffects of just the disulfide and just the double mutation (seeMethods). Because the Cys pairs that we analyzed were highlycross-linked before DTT and highly reduced after DTT, thecorrected effects were not very different from the originalobserved effects.

We also made the approximation of fitting a single Boltzmannfunction with a single V50 to the G–V curve for a mixture ofcross-linked and un-cross-linked subunits, which in reality was aweighted sum of at least two G–V curves. Similarly, we fittedsingle exponential functions with single rate constants to thetime courses of activation and of deactivation.

As a prerequisite to this approach, we showed that the onlyeffect of DTT on function was via the reduction of the disulfidebetween the substituted Cys. There was no effect of DTT on thefunction of pWT � or on the modulation of BK channel functionby pWT �1 (Fig. S4A). Thus, even though BK �1 has in itsextracellular loop four Cys that presumably form two disulfides,either these disulfides were not susceptible to reduction underthe conditions we used or their reduction did not affect thefunctional characteristics tested. By contrast, the perturbedfunction of disulfide-cross-linked Cys-substituted � and Cys-substituted �1 was invariably restored toward wild-type functionby DTT reduction (Fig. S4 B–D).

The separate effects of the disulfide and of the doublemutation on the V50 and on the rate constants for opening andclosing were calculated for 10 combinations of � and �1 Cysmutants, 4 of these forming disulfides between the flanks of S0and TM2, 4 forming disulfides between the flanks of S1 andTM1, and 2 forming disulfides between the flanks of S2 and TM1(Fig. 5). In all of these combinations except one, the effects ofthe substitution for the native residues of two Cys linked by adisulfide are larger than the effects of just the substitution of tworeduced Cys. The exception is � R17C and �1 Q155C. This is theonly pair tested in which the disulfide has little or no effect onany of the functional parameters. The cross-linking of all of theother pairs causes an increase in V50 by 40–130 mV (Fig. 5A), amoderate slowing of activation by up to a factor of 3 (Fig. 5B),and speeding of deactivation by a factor of up to 10 (Fig. 5C).Thus, each of these disulfides stabilizes the closed state of thechannel relative to the open state.

DiscussionDisulfide Cross-Linking. The induction of disulfide bond formationbetween substituted Cys has been widely used to infer localstructure and dynamics of proteins (26). We have not induceddisulfide bond formation but determined rather the extent ofendogenous disulfide-bond formation between pairs of substi-tuted Cys, one in � and one in �1. The Cys were substituted forat least the first four residues just extracellular to the membranedomains of the seven TM helices in BK � and of the two TMhelices in BK �1 (Fig. 1). Endogenous disulfide bond formation

can be due to spontaneous, uncatalyzed disulfide formation bydissolved O2 or to catalytic oxidation by protein disulfideisomerase (PDI) homologs in the endoplasmic reticulum (ER)(27), by secreted PDI homologs (28), or by secreted oxidases(29). We argued previously that the disulfide-bond formationbetween the juxtamembranous Cys that we observed in HEK293cells was due to PDI in the ER and, furthermore, that the extentof PDI-catalyzed disulfide bond formation reflects the proximityof the reduced Cys residues in a stable structure of the pro-tein (21).

Fig. 5. Functional effects of the replacement of native residues by cysteinesand their endogenous disulfide cross-linking. Ten Cys-substituted � and �1pairs were expressed in HEK cells. Recording was in outside-out macropatcheswith 10 �M Ca2� in the pipette. Black bars show the effects due to thedisulfide, and gray bars show the effects due to the double mutation. Effectson V50 (A), the rate constant for opening (B), and the rate constant for closing(C) were calculated as described in Methods.

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Locations of �1 TM1 and TM2 in a Model of BK �. From the extentsof disulfide bond formation, we have inferred the relativeproximities of substituted Cys in the flanks of the TM helices of� and �1. We extrapolate from these flanking residues to theextracellular ends of the membrane-embedded helices them-selves (Fig. 4). In the case of TM2 and S0, this extrapolation iscertainly correct because the Cys substituted for Arg-20, whichis the first residue just outside of the membrane in S0, was �80%cross-linked to the Cys substituted for Gln-155, the first residuejust outside the membrane in TM2 (Fig. 3B). In the case of TM1and S1, among the four TM1 flanking Cys tested, the Cys closestto the membrane, P40C, formed cross-links to the greatest extentwith Cys in the flank of S1. In S1, however, it was the N136C,three residues from the membrane, which most readily formeda cross-link with P40C. Similarly, among the pairs of Cys in TM1and S2 tested, the greatest extent of cross-linking was betweenTM1 Q43C, three residues from the membrane, and S2 F144C,four residues from the membrane. Thus, somewhat greaterflexibility and reach appear to be required to get extensivecross-linking between the flanks of TM1 and S1 or S2 thanbetween the flanks of TM2 and S0, consistent with the ends ofTM2 and S0 being closer than the ends of TM1 and S1 or S2.These results and the almost complete lack of cross-linking ofCys in the flanks of TM1 or TM2 to Cys in the flanks of S3–S6are the basis our placement of the extracellular ends of TM1 andTM2 in a model of a tetramer of BK � (Fig. 4).

The model of the BK � tetramer is itself based on a model ofthe closed state of Kv1.2 (30), on which we superimposed thelocation of the extracellular end of S0, which we previouslyinferred from the endogenous cross-linking of substituted Cys inthe flanks of S0–S6 (21). The pattern of cross-linking of the S0flank to the flanks of S1–S4 was the same in the presence andabsence of �1. The lines connecting S0 to the extracellular endsof S1, S2, and S3–S4 and the lines connecting �1 TM1 and TM2to � S0, S1, and S2 are color-coded to represent the averageextents of disulfide cross-linking, as given in Results (Fig. 4).

The positions of the extracellular ends of TM1 and TM2 arecompatible with previously reported �–� interactions. The N-terminal tail and S0 were proposed to include a docking site for�1 (8). More recently, we showed that �1 association with �required TM segments within the voltage-sensor domain, but notS5 or S6 (17). The extracellular ends of TM1 and TM2 begin andend the �1 extracellular loop. This loop affects the binding ofcharybdotoxin and iberiotoxin (13–15), the sites for which are inthe vicinity of the mouth of the pore (31). The distance in ourmodel from the ends of TM1 and TM2 to the mouth of the poreis �43 Å, which could be readily spanned by the 115-residueloop. The loops of the other �s are even longer.

We do not know the positions of the intracellular ends of TM1and TM2 in �1; these helices are unlikely to traverse themembrane normal to the plane. We have not yet mapped thepositions of the extracellular ends of �2–�4 TM helices and alsodo not know the positions of the intracellular ends of these TMhelices. If we assume that the �1 and �2 TM helices are locatedsimilarly, we should consider a constraint on the position of theintracellular end of �2 TM1. For the �2-subunit, which demon-strates N-type inactivation, a minimum of 12 residues are neededto allow the three N-terminal inactivating residues to reach thepore (32). Because 12 residues can span a maximum of 40 Å, thisconstrains the position of the intracellular end of TM1 in �2,certainly not inconsistent with our location of the extracellularend of TM1 in �1.

Functional Effects of Cross-Linking. Each of the Cys-substituted �and Cys-substituted �1 pairs that we tested was functional.Neither the substitution of two residues with Cys nor thedisulfide cross-linking of these residues completely disruptedvoltage-gating (Fig. 5). Based both on the high extents of

cross-linking and the functionality after cross-linking, these Cyswere close in a stable structure of the �–�1 complex. The pair� R17C (S0) and �1 Q155C (TM2), which was almost completelydisulfide cross-linked, had unperturbed function, consistent withthe tolerance of the channel structures in both the closed andopen states to this disulfide. Because �R17C (S0) also readilyformed a disulfide with �R201C in the short S3–S4 loop, whichalso did not perturb function (21), we conclude that the S0 flankis f lexible, and that locking it against either the S3–S4 loop oragainst the TM2 flank does not conflict with the functionaltransitions during activation and gating.

In all other pairs tested for function, cross-linking stabilizedthe closed state relative to the open state: V50 was significantlyincreased, the rate constant for opening was slowed by up to afactor of 3, and the rate constant for closing was increased by upto a factor of 10 (Fig. 5). One possibility is that when the disulfideis being formed in the ER, where we presume the membranepotential is zero and the [Ca2�]i is low, the voltage sensors aredeactivated and the channel is in the closed state. The proximi-ties of Cys that are being locked in by disulfide bond formationare those of the closed state structure. There is enough flexibilityin the flanks even after disulfide bond formation to permit theconformational changes concomitant with activation and gating,but the cross-links transmit tension to the activated voltagesensors, tending to return them to the deactivated state. Thecross-links, however, are responsible for only part of the func-tional effects, because the pair of reduced Cys themselvesstabilize the closed state, as seen in the effects on V50.

Implications for �1 Function. Compared with other voltage-gatedK� channels, the V50 for gating-charge movement of the voltagesensors of BK � alone is shifted to much more positive voltages,and even though the magnitude of the gating charge per voltagesensor is smaller (33–35), the electrostatic energy needed toactivate each voltage sensor is greater (21). We speculated thatthe association of S0, unique to BK �, with the S1–S4 voltage-sensor domain contributes to the stabilization of the closed staterelative to the open state (21). We have now found that �1 TM1and TM2 associate with the voltage-sensor domain of BK �, withTM1 close to S1 and S2 and TM2 close to S0. The effect of �1on the function of � is complex (18, 34, 36). At 0.5 nM Ca2�, �1decreases the V50 for gating-charge movement (34). Paradoxi-cally, at Ca2� concentrations �1 �M, �1 increases the V50 forchannel opening. At Ca2� concentrations �1 �M, however, �1decreases V50 for channel opening. At all Ca2� concentrations,�1 slows both channel opening and closing.

These modulating effects require that the contacts of �1 with� are different in the deactivated and activated states of thevoltage sensor. The different contacts are the basis for therelative stabilization by �1 of one state relative to another. Weinfer from the cross-linking results that the extracellular ends of�1 TM1 and TM2 are close to the extracellular ends of S0, S1,and S2 in the �-subunit voltage-sensor domain and, furthermore,that tying these ends together with disulfide bonds, presumablywhile the voltage sensor is in the deactivated state, stabilizes thedeactivated state relative to the activated state. It is clear,however, that none of the residues mutated to Cys in theextracellular flanks of the TM helices are irreplaceable for �1function and that �–�1 contacts involving these particularresidues are unlikely to play major functional roles. We do notknow whether or not contacts between the �1 TM helices and �S0, S1, and S2, assuming that contacts between these helicescontinue in the membrane domain, are different in the activatedand deactivated states of the voltage sensor. It is possible thatTM1 and TM2 merely serve to position the extracellular loopand the intracellular �1 N- and C-terminal tails, required for �1function (18, 19). On the other hand, it is also possible that thetails serve to position TM1 and TM2, which are positioned like

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pincers on the voltage-sensor domain. It will be a challenge toidentify all �–� interactions that are different in the differentfunctional states.

MethodsConstructs. The �1-subunit [KCNMB1; 191 residues; molecular weight (mw)21,700] was cloned by RT-PCR from murine smooth muscle RNA (Clontech).Mutants of �1 and of the BK �-subunit (mSlo1, KCNMA1, GenBank accessionno. NM�010610; 1,169 residues; mw 131,700) were generated by site-directedmutagenesis using QuikChange XL (Stratagene). Cys substitutions were madein a pWT �, in which Cys-14 and Cys-141 were mutated to Ala, and in pWT �1,in which Cys-18 and Cys-26 were mutated to Ala.

Expression, Surface Biotinylation, and Cell Lysis and Immunoprecipitation.HEK293 cells were cultured, transfected, surface-biotinylated, and solubilizedas described in refs. 17 and 21. For coimmunoprecipitation, see Fig. S2.

Quantitating �–� Cross-Linking. Surface-biotinylated BK channels were boundand eluted from Neutravidin-Sepharose beads (Pierce); one-half was reducedwith DTT, and one-half was not; both the unreduced and the reduced portionswere electrophoresed, immunoblotted, and quantitated as described in ref.21. We calculated the extent of cross-linking, from the band at 150 kDa dividedby the sum of the bands at 125 kDa and 150 kDa. In each experiment, duplicatelanes were run and the results averaged. All mutants were independently

expressed and tested twice if there was little cross-linking and three or moretimes if there was substantial cross-linking.

Functional Effects of Cross-Linking. G–V curves and the activation and deacti-vation kinetics were determined with inside-out and outside-out macropatchexperiments, as described in refs. 17 and 37. The effect on V50 was calculatedas �V50 � V50,MUT � V50,pWT. The effects on the rate constants of opening andclosing were calculated as log(kMUT/kpWT-HRV). The rationale is that V50 isproportional to the free energy between ground states, and log(k) is propor-tional to the free energy between a ground state and a transition state, andtherefore V50s can be added and subtracted, and the same applies to log(k)s.The separate effects of the disulfide bond and of the double mutation to Cyswere calculated from the effects before and after DTT, as described in SIMethods.

ACKNOWLEDGMENTS. We thank Vladimir Yarov-Yarovoy and William Cat-terall for supplying the Kv1.2 model used in Fig. 4, and Maria Garcia, GregoryKaczorowski, and Daniel Cox for comments on the manuscript. This work wassupported in part by National Heart, Lung, and Blood Institute/NationalInstitutes of Health (NIH) Grant P01 HL081172, National Institute of Neuro-logical Disorders and Stroke/NIH Grant R01 NS054946, and National Center forResearch Resources/NIH Grant UL1 RR024156, and by the Arlene and ArnoldGoldstein Family Foundation. S.O.M. is an Established Investigator of theAmerican Heart Association. R.S.W. has a Glorney–Raisbeck Fellowship fromthe New York Academy of Medicine.

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