Cortisone dissociates the Shaker family K+ channels fromtheir b subunitsYaping Pan1,2, Jun Weng1,2, Venkataraman Kabaleeswaran1, Huiguang Li1, Yu Cao1, Rahul C Bhosle1 &Ming Zhou1

The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential forcellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked toepisodic ataxia and atrial fibrillation. All Kv1 channels assemble with b subunits (Kvbs), and certain Kvbs, for example Kvb1, havean N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kvb1, althoughnever reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channelactivity by binding to Kvb1. A crystal structure of the Kvb-cortisone complex was solved to 1.82-A resolution and revealed novelcortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kvb. The new mode of channelmodulation may be explored by native or synthetic ligands to fine-tune cellular excitability.

Voltage-dependent potassium channels (Kv) are tetrameric integralmembrane proteins that, upon membrane depolarization, allowpotassium ions to flow out of the cell. The outflow of potassiumions brings the membrane potential back to the resting value andtherefore controls the timing and duration of the action potential. Inhumans, loss-of-function mutations in Kv1 channels are directlylinked to episodic ataxia and atrial fibrillation1,2. Native Kv1 channelsassemble with the cytosolic b subunit (Kvb), which is also a tetramer,to form a stable (Kv1)4(Kvb)4 complex that is preserved throughpurification3–6. The assembly of the Kv1-Kvb complex occurs in theendoplasmic reticulum7, and reciprocal immunocoprecipitationexperiments have shown that all Kv1 channels assemble with Kvb,and vice versa8.

There are three mammalian Kvb genes (those encoding Kvb1, Kvb2and Kvb3), and all have a highly conserved core domain (B330 aminoacids) with more than 80% sequence identity. The conserved cores ofKvb1 and Kvb2 are functional aldo-keto reductases (AKR) that useNADPH (1) as a cofactor9,10. In addition to the AKR core, Kvb1 hasan N-terminal segment that closes an open Kv1 channel by the N-typeinactivation mechanism11. In this mechanism, the N-terminal seg-ment, also called the inactivation gate, functions as a tethered channelblocker: it physically occludes the open channel pore within a fewmilliseconds after the channel is opened by membrane depolariza-tion12–16. In principle, releasing Kvb1 from the channel wouldpotentiate Kv1 current. However, dissociation of the Kv1-Kvb complexhas never been observed.

We have found that cortisone (2) binds to Kvb and increases Kv1current. Structural studies revealed two types of cortisone bindingsites on Kvb: one close to the NADPH cofactor and another at

the interface of the two neighboring Kvb subunits. Further studiesdemonstrated that only the interface binding site is required forchannel modulation, and that cortisone promotes dissociation ofKvb from the channel.

RESULTSCortisone potentiates Kv1.1 channel through Kvb1The conserved AKR core of Kvb co-purifies with an NADPH cofac-tor9,10,17, and we monitored the NADPH fluorescence to identify small-molecule compounds that bind to Kvb. Cortisone was identified bythis assay because it reduced the fluorescence by 21 ± 0.7% (n = 3,Fig. 1a,b). The change in fluorescence occurred immediately after cor-tisone was mixed with the protein and remained stable for the durationof the experiment (20 min). This effect on fluorescence intensity isdifferent from that induced by Kvb substrates, which oxidize theKvb-bound NADPH and therefore eliminate the fluorescence9,10.

To test whether cortisone modulates channel current, the Kv1.1channel was co-expressed with Kvb1 in Xenopus laevis oocytes andpotassium current was recorded on inside-out patches. The presenceof fast inactivation indicates that Kv1.1 and Kvb1 are co-assembled,because Kv1.1 expressed alone produces current that is not inactivat-ing (Fig. 1c,d). When 500 mM cortisone was perfused to theintracellular side of the channel, the onset rate of channel inactivationdecreased significantly from 302 ± 7 s–1 to 89 ± 4 s–1 (n ¼ 13,P o 0.001 in paired Student’s t-test), and increases of peak and steadystate currents were observed (Fig. 1c). The increase in current was dueto decreased onset rate of inactivation, because the rate of recoveryfrom inactivation remained essentially unchanged (13.9 ± 1.1 s–1

before and 12.0 ± 0.8 s–1 after cortisone perfusion, n¼ 4). The current

Received 22 July; accepted 2 September; published online 21 September 2008; doi:10.1038/nchembio.114

1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA.2These authors contributed equally to this work. Correspondence should be addressed to M.Z. (mz2140@columbia.edu).

70 8 VOLUME 4 NUMBER 11 NOVEMBER 2008 NATURE CHEMICAL BIOLOGY

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increase reached steady state in approximately 5 min and was notreversed even when perfused extensively with the normal insidesolution. To quantify the cortisone effect on current increase, wedefined DIss, the increase of the steady state current normalized to theinitial inactivating current. The cortisone effect was highly reprodu-cible, with a DIss of 84 ± 4% (n ¼ 13, Fig. 1e). In contrast, the vehiclecontrol (1% DMSO) induced a significantly smaller response, with aDIss of 10 ± 0.7% (n ¼ 8, P o 0.001 in ANOVA test, Fig. 1e,f). Tofurther characterize the cortisone effect, the DIss values were measuredat different cortisone concentrations and the data were well fit bya Hill equation with an effector concentration for half-maximumresponse (EC50) value of 46 ± 1.1 mM and a Hill coefficient of0.97 ± 0.09 (Fig. 1g).

To find out whether the cortisone effect is mediated by theconserved AKR core of Kvb1, we tested cortisone on a chimericchannel, Kv1.1-inact, which was constructed by connecting the first70 amino acid residues of Kvb1 directly to the N terminus of Kv1.1.Thus, Kv1.1-inact is a Kv1.1 channel that has an N-type inactivationgate from Kvb1 but does not have the conserved AKR core domain.Kv1.1-inact produced inactivating current similar to that from co-expression of Kv1.1 and Kvb1 (Fig. 1h), but the current was notpotentiated by cortisone. Cortisone (500 mM) induced a DIss of 6 ±0.8% (n ¼ 7, Fig. 1e,h) on Kv1.1-inact, which was not significantlydifferent from that of the vehicle control and significantly smallerthan that from co-expression of Kv1.1 and Kvb1. In addition, wealso examined cortisone on Kv1.1 expressed without Kvb1, and theDIss was 4.5 ± 0.5% (n ¼ 5, Fig. 1d,e). Thus, we conclude thatthe large potentiation of current by cortisone is mediated by theconserved core domain of Kvb1.

Two cortisone molecules bind to each Kvb subunitA structural approach was taken to determine where cortisonebinds on Kvb. The conserved AKR core of Kvb1 protein can beexpressed and purified in large quantity but is prone to aggrega-tion and did not yield crystals of sufficiently high diffractionquality even after extensive crystallization trials. Therefore theAKR core of Kvb2 was used for co-crystallization with cortisone.Given that the amino acid sequence of the Kvb1 core is B80%identical to that of Kvb2, the structures of the two are likelysimilar. Kvb2 was not used in the functional studies in the firstplace because it does not have the N-type inactivation gate. To testwhether the conserved core of Kvb2 also mediates the cortisoneeffect, we used a chimera, Kvb12, which was constructed bysplicing the inactivation gate of Kvb1 to the conserved core ofKvb2 (refs. 5,18). Similar to Kvb1, Kvb12 produced fast inactiva-tion when co-expressed with Kv1.1. More importantly, the inacti-vation was reduced by cortisone (500 mM) with a DIss of 89 ± 4%(n ¼ 12, Fig. 2a,b), which was significantly larger than that of thevehicle control (DIss ¼ 10 ± 0.8%, n ¼ 4; P o 0.001 in ANOVAtest, Fig. 2a,b) but not significantly different from that mediatedby Kvb1 (P 4 0.05 in ANOVA test, Fig. 2b).

Crystals of the Kvb2 core in complex with cortisone grew with atetragonal symmetry (I422), and the structure was solved bymolecular replacement. The structure refined well (Rfree ¼ 20.7%,Supplementary Table 1 online) using data measured to 1.82 ABragg spacing. A simulated annealing Fo – Fc (observed andcalculated structure factor) omit map revealed unambiguouslythe location and the molecular structure of two types of cortisonebinding sites for each Kvb subunit. One cortisone molecule binds

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Figure 1 Cortisone potentiates Kv1.1 current via the AKR core of Kvb. (a) Fluorescence spectra of Kvb2 protein before (0 min, black) and 1 and 5 min after

mixing with 500 mM cortisone (red). (b) Chemical structure of cortisone. The rings and carbon atoms are individually labeled. (c,d) Current traces recorded

on inside-out patches from oocytes injected with both Kv1.1 and Kvb1 (c) and with Kv1.1 (d) before (black) and after (red) perfusion of 500 mM cortisone.

(e) DIss after perfusion of cortisone or vehicle control. NS, not significantly different; ***P o 0.001, both by ANOVA. (f) Current traces recorded on inside-

out patches from oocytes injected with both Kv1.1 and Kvb1 before (black) and after (red) perfusion of vehicle control (1% DMSO). (g) Normalized DIssversus cortisone concentrations. The mean values of 3 to 13 patches are shown for each concentration. The solid curve is a Hill equation fit to the data

points. All error bars are s.e.m. (h) Current traces recorded on inside-out patches from oocytes injected with Kv1.1-inact before (black) and after (red)

perfusion of 500 mM cortisone.

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close to the NADP cofactor, and therefore we define the site asthe enzymatic site; another cortisone molecule binds in betweenthe two neighboring Kvb subunits, and we define it as the interfacesite (Fig. 2c).

Channel modulation does not require theenzymatic siteTo examine which binding site is responsiblefor the cortisone effect on channel current, weperturbed the binding site by making pointmutations and examined cortisone binding byX-ray crystallography. For mutations that

eliminated cortisone binding, we then investigated whether they stillmediated the cortisone effect on channel current.

At the enzymatic site, cortisone has hydrophobic interactions withthe side chain of residue Trp121: the A and B rings of cortisone stack

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Figure 3 Binding to the enzymatic site is not required for channel modulation. (a,b) Stereoview of the enzymatic site, for cortisone cocrystallized with the

wild-type (a) or the W121A mutant (b) Kvb2. The Fo – Fc omit maps (blue mesh), calculated with cortisone and the side chains of residues 189 and 121

omitted, were contoured at 2s level. The backbones are shown as green cartoon, NADP as cyan stick, amino acid side chains as stick, and cortisone as ball

and stick. For amino acid side chains and cortisone, carbon is shown in yellow, nitrogen in blue and oxygen in red. (c) Current traces of Kv1.1 co-expressed

with the W155A mutant Kvb1 before (black) and after (red) perfusion of either cortisone (500 mM, left panel) or vehicle control (1% DMSO, right panel).

(d) DIss after perfusion of cortisone or vehicle control. NS, not significantly different; ***P o 0.001, both by ANOVA. (e) Normalized DIss for different

cortisone concentrations in Kv1.1 co-expressed with the W155A Kvb1 (&) or Kv1.1 co-expressed with Kvb1 wild type (J). The mean values of 3 to

13 patches are shown for each concentration. All error bars are s.e.m.

Figure 2 Each Kvb2 binds two cortisone

molecules. (a) Current traces of Kv1.1 co-

expressed with Kvb12 before (black) and after

(red) perfusion of either cortisone (500 mM, left

panel) or vehicle control (1% DMSO, right panel).

(b) DIss after perfusion of cortisone or vehicle

control. NS, not significantly different; ***P o0.001, both by ANOVA. Error bars are s.e.m.

(c) Stereoview of tetrameric Kvb2 core in complex

with cortisone. The four-fold axis is perpendicular

to the plane of the paper, and its position is

marked (+). One pair of diagonally opposed Kvbsubunits are colored in light green, and another

pair in dark green. NADP is shown as cyan stick.

Cortisone is shown as a space-filled model withcarbon atoms colored in yellow and oxygen atoms

in red. The N and C termini are labeled as N

and C, respectively.

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against the flat surface of the indole ring (Fig. 3a). Therefore Trp121was mutated to alanine, the W121A mutant was co-crystallized withcortisone and the structure was solved to 2.0-A resolution (Rfree ¼24.0%, Supplementary Table 1). In the mutant structure, althoughcortisone density was clearly resolved at the interface site (Supple-mentary Fig. 1 online), no cortisone density was present at theenzymatic site (Fig. 3b). In addition, the Arg189 side chain con-formation provides further indication that a cortisone molecule is notpresent at the enzymatic site: when a cortisone molecule is present atthe enzymatic site, the Arg189 side chain adopts a bend conformation,as seen in the structure of Kvb2-cortisone (Fig. 3a). However, in theabsence of cortisone, the Arg189 side chain adopts an extendedconformation, as seen in the structure of Kvb2(W121A)-cortisone(Fig. 3b) and in that of the wild-type Kvb2 crystallized withoutcortisone (Supplementary Fig. 2 online). The overall structure ofcortisone in complex with the W121A mutant Kvb2 is similar to thatof the wild type (Protein Data Bank (PDB) ID 1EXB; ref. 5), with anr.m.s. deviation of 0.22 A for all the main chain atoms.

The equivalent position of Trp121 on Kvb1 is Trp155, and wemutated this residue to alanine. When Kv1.1 was co-expressed with

W155A Kvb1, inactivating current was observed, which indicates thatthe mutant Kvb1 had assembled with the channel (Fig. 3c). Cortisone(500 mM) induced a large increase in channel current, with a DIss of93 ± 8% (n ¼ 6, Fig. 3d), which was significantly higher than that ofthe vehicle control (DIss ¼ 13 ± 0.7%, n ¼ 4; P o 0.001 in ANOVAtest) but not significantly different from that mediated by the wild-type Kvb1 (P 4 0.05 in ANOVA test). To further investigate thecortisone effect mediated by the W155A mutant Kvb1, the DIss wasmeasured at different cortisone concentrations and the data were wellfit by a Hill equation, with an EC50 of 33 ± 1.1 mM and a Hillcoefficient of 0.96 ± 0.07, both similar to those of the wild-type Kvb1(Fig. 3e). Therefore binding of cortisone at the enzymatic site is notrequired for channel modulation.

The interface site is required for channel modulationAt the interface site, cortisone fits into a deep pocket formed by twoneighboring Kvb subunits (Fig. 4a,b). The A, B and C rings ofcortisone are immersed in the binding pocket and make extensivecontacts with residues lining the pocket (Fig. 4b). Compared withcortisone-Kvb interactions observed at the enzymatic site, more

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Figure 4 Binding to the interface site modulates channel inactivation. (a) Stereoview of an interface binding site

for the wild-type Kvb2-cortisone complex when the four-fold axis is perpendicular to the plane of the paper. Part

of the two neighboring Kvb2 subunits are shown as surface representation, colored in light and dark green.Cortisone is shown as ball and stick, with carbon in yellow and oxygen in red. (b,c) Stereoview of the interface

binding site, for cortisone cocrystallized with the wild-type (b) or the I211R mutant (c) Kvb2, both viewed with

the four-fold axis in parallel to the plane of the paper. The Fo – Fc omit map (blue mesh), calculated with

cortisone and the side chain of residue 211 omitted, was contoured at 2s level. The backbones of the two

neighboring Kvb subunits are shown as cartoon and colored in light and dark green. Select side chains are

shown as stick, and cortisone is shown as ball and stick, with the same color coding as in Figure 3. (d) Current

traces of Kv1.1 co-expressed with the V245R mutant Kvb1 before (black) and after (red) perfusion of cortisone

(500 mM, left panel) or vehicle control (1% DMSO, right panel). (e) DIss after perfusion of cortisone or vehicle

control. NS, not significantly different; ***P o 0.001, both by ANOVA. Error bars are s.e.m.

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residues participate in coordinating cortisone; some of them, forexample Arg203 and Glu167, are part of the interface between thetwo neighboring Kvbs. Using the structure as a guide, we analyzedin silico mutations to the residues lining the binding pocket, and wefound that mutating residue Ile211 to one with a bulkier side chain,such as arginine or tryptophan, may interfere with cortisone bindingand at the same time have a minimal impact on Kvb tetramerization.We therefore produced I211R and I211W mutant Kvb2s, co-crystal-lized the mutants with 2 mM cortisone and solved the structures to1.95 A and 1.9 A, respectively. Because the two mutations had thesame effect on cortisone binding, the I211R structure (Rfree ¼ 23.4%,Supplementary Table 1) is presented. In the wild-type structure theisoleucine side chain packs against the hydrophobic core of the protein(Fig. 4b), whereas in the mutant structure the arginine side chainprotrudes into the binding pocket and forms a steric hindrance toprevent cortisone from entering the binding pocket (Fig. 4c). As aresult, although cortisone density was observed at the enzymatic site(Supplementary Fig. 3 online), no cortisone density was present atthe interface site. The loss of cortisone binding at the interface site isdue to local perturbations at the binding site, because the structure ofKvb2(I211R)-cortisone aligns well with that of the wild-type Kvb2,with an r.m.s. deviation of 0.22 A.

The equivalent of the Ile211 on Kvb1 is a valine at position 245, andwe mutated this residue to arginine and co-expressed the V245R Kvb1with Kv1.1. Cortisone, even at 500 mM, only induced a small increaseof current, with a DIss of 13 ± 1% (n ¼ 13, Fig. 4d,e)—not

significantly different from that of the vehicle control (DIss ¼ 10 ± 1%,n ¼ 4; P 4 0.05 in ANOVA test); however, the DIss was significantlysmaller than that of the wild-type Kvb1 (P o 0.001 in ANOVA test).Thus, we conclude that cortisone binding at the interface site isrequired for channel modulation.

Cortisone compromises Kv1-Kvb assemblyExcept for small adjustments at the cortisone binding sites, the overallstructure of Kvb2-cortisone is almost identical to that of Kvb2, with ar.m.s. deviation of 0.21 A for all the main chain atoms. To furtherunderstand how cortisone binding induces an increase of current, wealigned Kvb2-cortisone onto the structure of the Kv1.2-Kvb2 complex(PDB ID 2A79; ref. 3).

The structures of Kvb2 in complex with either an entire Kv1channel3 or the intracellular tetramerization domain (T1) of thechannel5 both showed that Kvb docks onto the channel by interactingwith four highly conserved loops from the T1 domain. When theKvb2-cortisone structure was aligned with that of Kv1.2-Kvb2, itbecame clear that the interface site cortisone is close to where theT1 loops contact Kvb (Fig. 5a,b). The 16-position carbon, the17-position hydroxyl oxygen and the 21-position carbon of cortisoneare 2.9 to 3.7 A away from the side chains of Pro75 and Leu76 of theT1 loop (Fig. 5b). The proximity of cortisone to the T1 loopsnaturally led us to the hypothesis that cortisone may destabilize theKv1-Kvb complex. We examined this hypothesis with the followingtwo experiments.

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Figure 5 Cortisone promotes dissociation of Kvb. (a) The structure of Kv1.2 (magenta) in complex with Kvb2 (cyan) presented as cartoon (PDB ID 2A79).

Cortisone, shown in a space-fill model, was placed by aligning the Kvb2-cortisone structure with that of Kvb2 from PDB code 2A79. Dotted square

box demarcates the region magnified in b. (b) Stereoview of the Kv1-Kvb interface shown with side chains as stick and cortisone as ball and stick

representations. Distances (in A) between the atoms connected by the dotted lines are marked. In the Kvb2-cortisone complex, two neighboring Kvb subunits

are shown as dark and light greens, and in the Kv1-Kvb2 complex, the channel is shown as magenta and Kvb2 as cyan. (c) SDS-PAGE of the Kv1.1-T1 in

complex with Kvb2 wild type (left panel) or Kvb2(I211R) (right panel). On each gel, the molecular weight standard is the leftmost lane and labeled. For

other lanes, the higher molecular weight band is Kvb2, and the lower one is the T1 domain. Cortisone concentrations are marked on the top of each lane.

Ctr, 1% DMSO vehicle control. (d) Normalized Kvb2 intensity plotted versus cortisone concentrations. Error bars are s.e.m. (n ¼ 4). The solid curve is a Hill

function fit to the data points with an EC50 of 68 ± 1.1 mM, and a Hill coefficient of 2.9 ± 0.4. (e) Current traces of Kvb1-Kv1.1 connected chimera before

(black) and after (red) perfusion of cortisone (500 mM, left panel) or vehicle control (1% DMSO, right panel). (f) DIss after perfusion of cortisone or vehicle

control. NS, not significantly different; ***P o 0.001, both by ANOVA. Error bars are s.e.m.

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First, we co-expressed and purified the T1-Kvb2 complex and useda solid-phase binding assay to examine whether cortisone affects theassociation of the two. When the complex was incubated withcortisone, dissociation occurred with an EC50 value of 68 ± 1.1 mM(Fig. 5c,d). As a control, we expressed the T1 domain in complexwith the I211R mutant Kvb2, and we found that the mutant Kvb2did not dissociate from the T1 domain in the presence of 2 mMcortisone (Fig. 5c).

Second, we constructed a chimera, Kvb1-Kv1.1, by covalentlylinking the C terminus of Kvb1 to the N terminus of Kv1.1 so thatKvb1 was not able to diffuse away even when it fell off the T1 domain.The Kvb1-Kv1.1 chimera expressed well, and cortisone at 500 mMinduced a small change in channel current, with a DIss of 13 ± 1%(n ¼ 9, Fig. 5e,f), which was not significantly different from that ofthe vehicle control (DIss ¼ 10 ± 0.7%, n ¼ 4; P 4 0.05 in ANOVAtest). Results from both experiments support the hypothesis thatdissociation of Kvb1 from the channel is required for potentiationof channel current by cortisone.

DISCUSSIONIn conclusion, we have identified a binding site on Kvb through whichthe assembly of the Kv1-Kvb complex can be tuned. Dissociation ofKvb provides a new mechanism for modulation of Kv1 channelfunctions, and has the following three implications for potassiumchannel physiology and pharmacology.

First, acute dissociation of Kvb provides a new method to examineKvb’s physiological functions. The conserved core of Kvb is afunctional aldo-keto reductase, and oxidation of the Kvb-boundNADPH modulates channel activity9,10. Based on these observations,it has been proposed that Kvb is a redox sensor that couplesintracellular redox chemistry to the excitability of the cell. However,this hypothesis has not been tested in vivo. In addition to being afunctional aldo-keto reductase, Kvb increases surface expression levelsof Kv1 channels19,20 and is required for axonal targeting of Kv1channels in mammalian neurons21. Thus, eliminating Kvb in anorganism affects channel distribution and localization, making itdifficult to pinpoint the exact physiological functions of Kvb as anenzyme. The dissociation of Kvb in situ after the Kv1-Kvb complex hasalready been delivered to its proper cellular location will assist theunderstanding of Kvb’s physiological role.

Second, the observation that Kvb can be competed off the channelwith a small-molecule compound suggests that the Kv1-Kvb complexmay not be permanent. As a recent review has pointed out22, it is ingeneral very difficult to obtain small-molecule compounds that inter-fere with protein-protein interactions because the interfaces are usuallylarge and extensive, and do not have grooves and pockets for smallligands. In contrast, the Kv1-Kvb interface lacks an extensive interactionsurface3,5 (Fig. 5b) and, as this study has shown, has in fact a deeppocket suitable for a small ligand. These properties suggest that asso-ciation of Kvb to the channel is tunable under physiological conditions.The dynamic modulation of Kv1 channel activities by association ofKvb may be important for proper cellular responses to the differentredox environments that mammals experience during development.

Third, dissociation of Kvb1 potentiates channel current owing to lossof the N-type inactivation. Cortisone is therefore a rare Kv1 channelopener, and the interface cortisone binding site could be exploited bysmall-molecule compounds to modulate Kv1 channels. Because Kvbassembles exclusively with Kv1-family channels5,23,24, compoundstargeting Kvb should be highly specific to only the Kv1 channels.

Discovery of cortisone as a channel modulator was a coincidence:cortisone was identified from a small collection of B120 known

aldo-keto reductase substrates by a ‘‘low-throughput’’ manual screen,and the readout for the screen was Kvb-bound NADPH fluorescence(Fig. 1a). Although cortisone changes the fluorescence, it is not a Kvbsubstrate, and the interface binding site is far away from the active site.The micromolar-range EC50 value indicates that cortisone does notmodulate channel activity under normal physiological conditions. Theaccidental discovery of cortisone as a channel modulator, however,provides proof of concept for using a high-throughput screen toobtain small-molecule compounds that modulate association of Kvbto the channel. Furthermore, the interface binding site is largelyhydrophobic and recognizes the A, B and C rings of cortisone. It islikely that other members of the corticosteroid family, or theirsynthetic derivatives, may bind to the interface site as well. Thecombination of unbiased high-throughput screens and structure-based modification of cortisone promises higher affinity probes forachieving pharmacological control of Kv1-Kvb assembly.

METHODSFluorescence measurement of cortisone binding. Kvb2 conserved core

(residues 36–367) was expressed and purified as described9. The Kvb2-bound

NADPH fluorescence spectrum was recorded at room temperature (20–22 1C)

with an excitation wavelength of 360 nm at 1-nm slit size. 150 ml of purified

Kvb2 core (2 mM) in buffer A was used in each experiment. Buffer A contains

20 mM Tris (pH 8.0) and 150 mM KCl. After a spectrum was recorded, 3 ml of

cortisone stock solution (100 mM in DMSO) was pipetted into the Kvb2

solution and quickly mixed. A spectrum was recorded immediately after

mixture (time 0) and then at different time points afterwards. As a control

experiment, Kvb2 was substituted with free NADPH, and cortisone did not

induce a significant change in fluorescence.

Kvb2 crystallization. Purified Kvb2 protein was loaded onto a Superdex 200

column (GE Healthcare) for final purification. Kvb2 protein was concentrated

to B12 mg ml–1 and then mixed with 2 mM cortisone for co-crystallization.

Crystals were grown by the sitting-drop vapor diffusion method at 20 1C by

mixing equal volumes of protein with a reservoir solution containing 6–15%

glycerol, 1.5 M ammonium sulfate, 0.1 M Tris (pH 7.9–8.8). Glycerol

concentration in the reservoir solution was gradually raised to 25% for

cryoprotection, and crystals were flash-frozen in liquid nitrogen–cooled liquid

propane. Two crystal forms were obtained under similar conditions, one with a

P21212 symmetry and another one with an I422 symmetry. As the I422 crystal

form consistently gave better resolution, it was used for structural solution.

X-ray diffraction data collection and crystallography. For the Kvb2-cortisone

complex, X-ray diffraction data were collected from a frozen crystal on

Brookhaven National Laboratory (BNL) beamline X29. The intensity data

from a single crystal were integrated and scaled using the HKL2000 program

suite25. Molecular replacement solution was obtained using the program

MOLREP26 starting with a model of Kvb2 (extracted from PDB ID 1EXB;

ref. 5), from which all solvent molecules and the cofactor NADP were removed.

The initial calculations, including rigid-body refinement and simulated anneal-

ing procedures, were performed using the program suite CNS27. Initial electron

density maps were calculated, and a difference map showed clear density for the

cofactor NADP and two cortisone molecules. NADP and cortisone were then

placed into the density. Cortisone was built with PRODRG28.

The model was adjusted in the program O29 against the 2Fo – Fc and Fo – Fc

maps, where Fo and Fc are the observed and calculated structure factors,

respectively. Water molecules were added depending on their location in

relation to significant electron density and the presence of satisfactory hydrogen

bonding interactions. The resultant models were then subjected to cycles of

refinement in which simulated annealing, coordinate minimization and

B-factor refinement were conducted in CNS. Model validation was conducted

using PROCHECK30, and structure alignment was performed with LSQKAB31.

Datasets for Kvb2(W121A)-cortisone and Kvb2(I211R)-cortisone were

collected at BNL beamlines X4A and X4C, respectively. Because both mutations

have the same I422 symmetry as the wild type, the refined wild-type

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Kvb2 model with only the protein atoms was used to calculate the initial

electron density map. In each case, the mutant side chain, the NADP cofactor

and one cortisone molecule were identified unambiguously by the electron

density map. The models were then further refined following the same

procedure as for the wild type. The structures shown in Figures 2–5 were all

drawn in PyMOL (http://pymol.sourceforge.net/).

Dissociation assay for the T1-Kvb2 complex. The Kv1.1T1-Kvb2 complex was

produced following a published strategy5. Briefly, the complementary DNA for

the T1 domain of Kv1.1 (residues 2–135) was cloned (between the NotI and SalI

sites) into a modified pET-SUMO vector (Invitrogen Inc.), which has a

kanamycin resistance gene. The expressed fusion protein has an N terminus

His6 tag, followed by a SUMO protein and the T1 domain. The histidine tag

and the SUMO protein are cleaved when the fusion protein is treated with

SUMO protease, which was produced in the lab. The conserved core of rat

Kvb2 (residues 36–367) was cloned (between the NdeI and XhoI sites) into a

modified pET31 vector, which has an ampicillin resistance gene, and the

expressed Kvb2 core did not have an affinity tag. Kv1.1T1-Kvb2 complex

protein was purified by cobalt affinity resin. For measuring dissociation, 20 ml

of cobalt resin, which absorbed B40 mg of T1-Kvb2 complex, was resuspended

in 1 ml of buffer B, supplemented with the desired amount of cortisone. Buffer

B contains 20 mM Tris (pH 8.0), 150 mM KCl, 1 mM b-mercaptoethanol and

10% glycerol by volume. After gentle mixing at room temperature for 30 min,

the resin was spun down and washed 4 times, each time with 1 ml of buffer B.

The resin was then resuspended in 200 ml of buffer B with 10 mg of SUMO

protease and incubated at room temperature for 30 min with gentle mixing.

The supernatant was collected, and 15 ml was loaded onto an SDS-PAGE for

further analysis. Each sample was loaded in duplicates as an internal control for

consistency of sample loading.

SDS-PAGE gels were stained with Coomassie Blue and digitized by the

FluorChem 8900 system (Alpha Innotech Co.) for quantification. The

intensities from the duplicates were averaged for each experiment. The

intensity of the T1 band, which varied o10% from lane to lane on each gel,

serves as a standard for the amount of protein loaded. The intensity of the

Kvb2 band was then normalized to its accompanying T1 band and plotted

versus cortisone concentration, and the data points were fit with a dose-

response equation.

Additional methods. The following methodologies can be found in Supple-

mentary Methods online: molecular biology, electrophysiological recordings

and co-expression of the Kv1.1T1-Kvb2 complex.

Data statistics. The Origin 7.5 software package (OriginLab) was used for

statistical analysis of the data. The results are expressed as mean ± s.e.m.

Student’s t-test and one-way analysis of variance (ANOVA) were used to assess

changes of a mean value.

Accession codes. Protein Data Bank: coordinates and structure factors for

Kvb2-cortisone, Kvb2(W121A)-cortisone and Kvb2(I211R)-cortisone have

been deposited under accession codes 3EAU, 3EB3 and 3EB4, respectively.

Coordinates of T1-Kvb2 complex5 (1EXB) and Kv1.2-Kvb2 complex3

(2A79) were deposited as part of previous studies. GenBank: Rattus

norvegicus Kv1.1, NM_173095; R. norvegicus Kvb2, CAA54142; R. norvegicus

Kvb1, NM_017303.

Note: Supplementary information and chemical compound information is available onthe Nature Chemical Biology website.

ACKNOWLEDGMENTSWe thank R. MacKinnon (Rockefeller University) for advice and generous helpthroughout the project. We thank C. Deutsch (University of Pennsylvania) andC. Miller (Brandeis University) for critical comments on the manuscript. Datafor this study were measured at beamlines X4A, X4C and X29 of the NationalSynchrotron Light Source. We thank J. Schwanof, R. Abramowitz, S. Myers,N. Whalen and R. Jackimowicz for technical support during data collection. Thiswork was supported by the American Heart Association (0630148N to M.Z. and0826067D to Y.P.), the US National Institutes of Health (HL086392 to M.Z.), theMarch of Dimes Birth Defects Foundation (research grant #5-FY06-20 to M.Z.)and a grant from the Pew Scholars Program (to M.Z.).

Published online at http://www.nature.com/naturechemicalbiology/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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