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Biophysical Journal

Volume 107

December 2014

2786–2796

Article Identification of a Cholesterol-Binding Pocket in Inward Rectifier KD (Kir) Channels Oliver Fu¨rst,1 Colin G. Nichols,2 Guillaume Lamoureux,3 and Nazzareno D’Avanzo1,* De´partement de Physiologie Mole´culaire et Inte´grative and Groupe d’E´tude des Prote´ines Membranaires (GE´PROM), Universite´ de Montre´al, Montreal, Quebec, Canada; 2Department of Cell Biology and Physiology and Center for Investigation of Membrane Excitabiltiy Diseases, Washington University School of Medicine, St. Louis, Missouri; and 3Department of Chemistry and Biochemistry, Centre for Research in Molecular Modeling (CERMM), Groupe d’E´tude des Prote´ines Membranaires (GE´PROM), Concordia University, Montreal, Quebec, Canada 1

ABSTRACT Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier Kþ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket.

INTRODUCTION Inward rectifier potassium (Kir) channels selectively control the permeation of Kþ ions across cell membranes with key roles in establishing the resting membrane potential of excitable cells, renal Kþ transport, regulation of pancreatic insulin secretion, and regulation of pacing in cardiomyocytes and neurons (1–7). Cholesterol, the major sterol component of all mammalian plasma membranes, modulates the function of various ion channels, including potassium (Kþ) channels (8–12), voltage-gated calcium (Cav) channels (13–17), voltage-gated sodium (Nav) channels (18,19), and chloride (Cl) channels (20,21). Cholesterol suppression of Kir currents was first observed in aortic endothelial cells (12), and affirmed by heterologous expression in Chinese hamster ovary (CHO) cells (11,22–24). Shortly thereafter, experiments in purified bacterial KirBac1.1 and KirBac3.1 (25–27) as well as human Kir2.1 channels (25) reconstituted into proteoliposomes confirmed that cholesterol regulation of Kir channels was independent of any other proteins or intracellular signaling pathways. Lack of inhibition by enantiomeric cholesterol (ent-cholesterol)—which has the same effects on membrane properties as natural cholesterol (28– 30)—in proteoliposomes containing purified channel proteins, indicates that the cholesterol action on prokaryotic KirBac1.1 and KirBac3.1 channels, as well as on human

Submitted June 6, 2014, and accepted for publication October 8, 2014. *Correspondence: [email protected] Editor: Chris Lingle. Ó 2014 by the Biophysical Society 0006-3495/14/12/2786/11 $2.00

Kir2.1 channels, results directly from enantioselective binding to the channel protein, and not from an indirect effect on lipid bilayer properties (25). Patch-clamp analysis suggests that cholesterol may reduce channel activity by locking the channels into prolonged closed states (11,23,25). Recent advances in x-ray crystallography have provided atomic structures of KirBac1.1 channels (31,32) and Kir2.2 channels (33). The latter share >72% sequence identity in the transmembrane region to human Kir2.1 channels (see Fig. 6) and are similarly regulated by cholesterol (21). With our previous findings regarding the lack of functional inhibition by ent-cholesterol in mind, we examined putative binding sites identified by computational docking calculations of cholesterol versus ent-cholesterol to the atomic structures of Kir2.2 and KirBac1.1 channels. MATERIALS AND METHODS Cholesterol docking calculations Cholesterol and ent-cholesterol structures and charge information were kindly provided to us by Dr. Brett Olson (Washington University in St. Louis, MO) (34). Sequence alignment for the generation of the Kir7.1 model was performed using the ClustalW webserver (35) (see Fig. 6). The homology model of Kir7.1 was generated by sequence threading and side chains were optimized to remove any putative steric clashes using ICM-Pro v.3.5.0-a (Molsoft LLC) (36). PDBQT structure files, containing charges and atom types were generated from the Protein Data Bank (PDB) files of Kir2.2 (PDB: 3SPI), KirBac1.1 (PDB: 2WLL), and Kir7.1 channel atomic models beginning with protonation, assignment of Gasteiger charges, merging of nonpolar hydrogen atoms to the bonded carbon

http://dx.doi.org/10.1016/j.bpj.2014.10.066

Cholesterol-Binding Pocket in Kir Channels atoms, and assignment of atom types using AutodockTools (ADT) 1.5.4 (37). Partial charges and rotatable bonds were also assigned to the cholesterol and ent-cholesterol ligands using ADT. Because ADT limits the number of rotatable bonds that can be sampled, rotatable bonds were limited to the ligands, and the receptor side chains were kept rigid following optimization. Docking of these ligands to single snapshots of the channels were confined to the transmembrane regions (including the slide helices) of subunits A and B, which would contain all interfaces found in the tetramer, and ˚ . Following 250 indepenwere performed using a default grid size of 0.36 A dent docking calculations for each ligand to each Kir channel structure, poses were clustered according to localization and orientation. Only poses in which the hydroxyl group of the ligand was directed to the intracellular or extracellular side of the channel, and where sterol moiety was entirely found in one-half or the other of the transmembrane (TM) domains (i.e., found where we expect the extracellular or intracellular leaflets to be) were considered acceptable. Putative binding pockets were identified by comparing clusters in which cholesterol binds with high frequency (quantified by % of poses) and ent-cholesterol either did not bind or bound with higher binding energy/lower affinity as estimated by Autodock. Stability of the highest frequency docking poses was assessed using molecular dynamics (MD) simulations. Four cholesterol molecules were simulated at once, by placing three additional molecules at symmetric positions around the Kir2.2 tetramer. Using the CHARMM-GUI server (38,39), each protein-cholesterol complex was embedded in a hexagonal simulation cell containing an explicit 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membrane and a 150 mM KCl solution. Each simulation system contains the four Kir2.2 monomers (from Cys-41 to Ser-372), four cholesterol molecules, 115 Kþ ions, 91 Cl ions, between 245 and 247 POPC molecules, and between 31,750 and 31,973 water molecules. Titratable residues Asp, Glu, and His were assigned default protonation states, except His-227, which was protonated on Nε instead of Nd. Cys-41 and Ser-372 residues were capped with neutral groups. Systems were simulated in the NPAT ensemble, at T ¼ 303.15 K (30 C) and p ¼ 1 atm, using a 2-fs time step and keeping all bonds involving H atoms at constant length. Standard CHARMM cutoff schemes and periodic boundary conditions were used. After 0.675 ns of MD equilibration during which all components were gradually allowed to move freely, 100 ns of MD production were performed. NAMD 2.9 (40) was used for all simulations.

Electrophysiology of Kir2.1 channels CHO-K1 cells were incubated at 37 C, 5% CO2 in F12 media (Sigma-Aldrich, Oakville, ON), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco BRL, Carlsbad, CA). Cells were plated on glass slides in a 60 mm dish and transfected with 4 mg of the appropriate Kir2.1 construct plus 1 mg of pEGFP cDNA using Lipofectamine 2000 in serum-free Optimem I media (Life Technologies, Waltham, MA) following the supplied protocol. 24–48 h after transfection, cells were washed with phosphate buffered saline (PBS), and incubated with 5 mM methyl-betacyclodextrin (MbCD) in Optimem I media for 4 h. Cells were washed again with PBS and incubated in Optimem I for an additional 4 h before use. Control cells underwent the same treatment in the absence of MbCD in the medium. S95H, I171L, and S95H/I171L mutations in Kir2.1 channels were made using Quikchange II mutagenesis kits (Stratagene, Santa Clara, CA) and sequenced for fidelity. Patch-clamp recordings were made in cell-attached mode for wild-type (WT), I171L, and S95H/I171L channels or whole-cell mode for S95H channels with pipette and buffer solutions containing 150 mM KCl, 10 mM HEPES, 2 mM MgCl2, 1 mM EGTA pH ¼ 7.4. Membrane patches were voltage-clamped using a HEKA UPC800 amplifier with matching head-stage and digitizer board (MDS Analytical Technologies, Toronto, Ontario, Canada). Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, Saint-Laurent, Quebec, Canada) to a resistance of ~1–3 MU and the data were digitized at a sampling rate of 10 kHz, and a low-pass analog filter was set to 1 kHz using GenPulse software (Michael

2787 Pusch, Instituto di Biofisica, Genova). Slope conductances were determined using the pClamp 10 software suite (MDS Analytical Technologies) and the remaining data analysis was performed using Origin7.0 (OriginLab, Northampton, MA).

Statistical analysis Statistical significance was analyzed using an unpaired t-test, and statistical significance (P < 0.05) is indicated by an asterisk.

RESULTS Docking calculations of cholesterol to KirBac1.1 and Kir2.2 channels Cholesterol and ent-cholesterol were docked to atomic structures of KirBac1.1 (PDB: 2WLL) and Kir2.2 (PDB: 3SPI) using Autodock 4.2 software. Following 250 docking calculations for each ligand, poses were assessed for orientation; only poses in which the hydroxyl group of the ligand was directed to the intracellular or extracellular side of the channel were considered acceptable. In total, 88 poses (35.2%) and 36 poses (14.4%) were accepted for KirBac1.1 docked to cholesterol and ent-cholesterol, respectively. For Kir2.2 channels, 184 (73.6%) cholesterol poses, but only 96 (38.4%) ent-cholesterol poses were acceptable. Accepted poses were then clustered according to orientation (both direction of hydroxyl group, and presentation of the rough face of cholesterol). Cholesterol docked to KirBac1.1 could be grouped into five clusters (Fig. 1), whereas for Kir2.2 channels, cholesterol docked within five clusters (Fig. 2). Given that ent-cholesterol is without inhibitory effect on KirBac or eukaryotic Kir channel activity, implicating direct protein-sterol interactions in channel inhibition, we anticipated that the most physiologically relevant cluster of poses would satisfy the following criteria: 1) cholesterol should bind to the channels with relatively high frequency (quantified by fraction of poses) and 2) ent-cholesterol either should not bind, or bind with significantly higher binding energy. Of the six clusters for KirBac1.1, only one site satisfied both conditions, a site to which cholesterol bound in 16.5% of poses, with relatively low energy (DG ¼ 6.91 5 0.37 kcal/mol), and to which ent-cholesterol bound with significantly lower affinity/higher energy (DG ¼ 5.95 5 0.52 kcal/mol) (Fig. 1), consistent with at least a fivefold decrease in affinity for ent-cholesterol. There was a single cluster in which ent-cholesterol did not bind, but this was not further considered because both 1) the affinity for this cluster and 2) the number of poses within the cluster, were low (5 of 91 accepted poses, respectively), indicating these are not preferred confirmations for the ligand. In the case of Kir2.2 channels, again only one cluster of poses had a high number of poses (82.6%) with low binding energy (DG ¼ 6.53 5 0.40 kcal/mol), whereas ent-cholesterol was not observed to bind in this location (Fig. 2). Biophysical Journal 107(12) 2786–2796

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A

B

Docking of ent-cholesterol to the KirBac1.1 structure revealed a similar location to that occupied by cholesterol, but dissimilar orientation, with the molecule rotated ~90 , and the carbon tail flipped into an alternate groove, lined by the residues F132, V133, S136, and G137 (Fig. 3 A). Such a groove is not available in Kir2.2 channels, and in other eukaryotic Kir channels it is occluded by a highly conserved phenylalanine residue (F175 in Kir2.2) (see Fig. 3 C). A forced overlay of ent-cholesterol in the preferred cholesterol orientation reveals steric clashes between the B ring of the ligand and residue L144 in KirBac1.1 and M70 in Kir2.2

FIGURE 1 Computational assessment of cholesterol binding in KirBac1.1 channels. (A) Putative docking sites of cholesterol and ent-cholesterol to KirBac1.1 (PDB: 2WLL). A representative pose from each cluster of docking sites is shown in different colors. (B) A cross-comparison of docked clusters of cholesterol and ent-cholesterol and corresponding energy (DG), estimated inhibition constant (Ki), and percent of total valid poses contained within that cluster. Only poses within one putative cluster had a high frequency of poses containing cholesterol, and a drastically reduced affinity for ent-cholesterol within the same region (highlighted in red box). To see this figure in color, go online.

(Fig. 3, B and C). This provides further consistency with this pocket being the biophysically relevant binding pocket for cholesterol and a molecular basis for lack of interaction with ent-cholesterol in Kir channels. MD simulations of cholesterol bound to Kir2.2 channel The 5 Kir2.2 binding poses illustrated in Fig. 2 A were simulated using MD. The time evolution of each of the four identical (but independently moving) cholesterol molecules was

A

B

Biophysical Journal 107(12) 2786–2796

FIGURE 2 Computational assessment of cholesterol binding in Kir2.2 channels. (A) Putative docking sites of cholesterol and ent-cholesterol to Kir2.2 (PDB: 3SPI). A representative pose from each cluster of docking sites is shown in different colors. (B) A cross-comparison of docked clusters of cholesterol and ent-cholesterol and corresponding energy (DG), estimated inhibition constant (Ki), and percent of total valid poses contained within that cluster. Only poses within one putative cluster had a high frequency of poses containing cholesterol, and a drastically reduced affinity for ent-cholesterol within the same region (highlighted in red box). To see this figure in color, go online.

Cholesterol-Binding Pocket in Kir Channels

FIGURE 3 Comparison of cholesterol and ent-cholesterol binding to KirBac1.1 and Kir2.2 channels. (A) A representative pose of cholesterol (orange) and ent-cholesterol (gray) docked to KirBac1.1 channels. Entcholesterol does not bind in the identical confirmation as cholesterol, but rather is rotated ~90 within the binding pocket. Furthermore, the acyl tail appears to favor an alternative groove. (B) An overlay of ent-cholesterol (gray) at this putative cholesterol (orange) docking site suggests ent-cholesterol cannot bind to KirBac1.1 channels at this site due to a steric clash with L144 and the B ring of the ligand. (C) An overlay of ent-cholesterol (gray) at this putative cholesterol (red) docking site suggests ent-cholesterol cannot bind to Kir2.2 channels at this site due to a steric clash with M70 and the B ring of the ligand. To see this figure in color, go online.

monitored by computing the distribution of the oxygen atom of the hydroxyl group and of the tertiary carbon atom of the tail (Fig. 4). Spatial distributions are calculated in 10-ns blocks, using 500 MD frames per block (one every 20 ps) ˚ 1A ˚  and binning the atom positions on a grid of 1 A ˚ cubic elements. To correct for random diffusion and 1 A rolling of the protein during the simulation, all frames are aligned to the Ca atoms of the original Kir2.2 crystal

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FIGURE 4 Time evolution of the position of cholesterol molecules docked to the putative binding pocket of Kir2.2 channel. (A) Spatial distributions of the cholesterol molecule hydroxyl oxygen atom and the tertiary carbon atom in the acyl tail, for each of the five docking poses presented in Fig. 2A (color-coded accordingly). Atoms are located inside the white surfaces during the first 10 ns of simulation and inside the deep blue/purple/orange/green/red surfaces during the last 10 ns of simulation (90 ns later). The starting poses for each cluster are shown in white and the final poses at the end of the simulation are shown in the cluster’s respective color. To see this figure in color, go online.

˚3 structure. Isosurfaces are drawn at the two counts/(1-A element)/(500 frames) level. The color saturation of the isosurface indicates the block used: white is the first 10-ns block (from 0 to 10 ns) and blue/purple/orange/green/red (consistent with poses in Fig. 2) is the last 10-ns block (from 90 to 100 ns). These simulations indicate that cholesterol oriented as it is in the red cluster (rough face pointed inward, toward the protein) is most stable. Similarly, poses from the orange cluster (rough face also pointed inward) seem to converge to those from the red cluster, but without the tails going as deep into the TM1 and TM2 cleft. Biophysical Journal 107(12) 2786–2796

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Cholesterol molecules from the blue cluster (rough face pointed outward) bind similarly to cholesterol molecules from the red cluster (although not as consistently), whereas cholesterol molecules from the green cluster are very mobile and do not adopt a consistent pose. From the purple cluster, 2 of the 4 cholesterol molecules slide away and align with the TM domains. Because stability within a pocket is indicative of binding affinity, these data provide support for cholesterol binding in the location and orientation of the red cluster identified by docking calculations. Characterization of the putative cholesterolbinding pocket in KirBac1.1 and Kir2.2 We further characterized this putative binding pocket by ˚ of the cholesterol examining which residues are within 5 A ligand for each pose within the cluster. The residues lining this binding pocket were identified quantitatively as those ˚ limit for >45% of the poses within this cluswithin this 5 A ter (Fig. 5). In this position, cholesterol is wedged between TM1 and TM2, with the hydroxyl group also interacting with the slide helix in the N-terminal direction. Interestingly, the predicted binding pocket is quite similar in KirBac1.1 and Kir2.2, although cholesterol is rotated nearly 180 and appears to bind more deeply into the pocket in KirBac1.1 (Fig. 5 C). This is apparently the result of a Phe residue that is in a different rotamer position than in the Kir2.2 structure (Fig. 5 C), and which may act as a gatekeeper for opening of a fenestration in the channel surface, enabling cholesterol to bind deeper within the channel. As a result, the hydroxyl group of cholesterol bound to Kir2.2 interacts with one residue (M184) of the neighboring side-chains TM2 (Fig. 5 B), whereas in KirBac1.1, cholesterol interacts only within a subunit (Fig. 5 A). Experimental confirmation of the putative cholesterol-binding pocket To experimentally test the putative cholesterol-binding pocket, we turned to mutagenesis and electrophysiological experiments in eukaryotic Kir channels. Because cholesterol is a hydrocarbon, protein-sterol interactions are not likely to be dependent on the hydrogen bond or electrostatic interactions, but rather on much weaker forces. Thus, predicting which mutation to generate is less straightforward. Kir7.1 channels have been shown to be unaffected by cholesterol depletion (22) and are therefore presumed to be insensitive to inhibition by cholesterol. To guide mutagenesis, we first examined cholesterol docking to a Kir7.1 homology model (see Figs. 7 and 8) based on the sequence alignment in Fig. 6. When cholesterol was placed in a space-filling model of Kir7.1 channels in the putative binding location in Kir2.2, the carbon tail sterically clashed with residues H67 and L148 (Fig. 7 A). Kir7.1 is the only member of the Kir family that carry that pair of residues (Fig. 6). In Biophysical Journal 107(12) 2786–2796

FIGURE 5 Determination of residues that form the putative cholesterolbinding pocket in KirBac1.1 and Kir2.2 channels. A representative pose of cholesterol docked to KirBac1.1 (A) and Kir2.2 (B) channels (left). The fre˚ of cholesterol for each pose within the cluster quency of residues within 5 A was assessed and is indicated as a percentage (right). Residues that interact with cholesterol with a frequency >45% of poses are highlighted in the structure. (C) (Left) An overlay of cholesterol bound to KirBac1.1 (cyan ribbon; orange cholesterol) and Kir2.2 (green ribbon; red cholesterol) channels. The binding pocket appears to be relatively conserved spatially, however, cholesterol appears to dock deeper into the channel protein, and with the rough face rotated nearly 180 . (Right) Closer examination of the crystal structures of these proteins indicate a phenylalanine that may act as a gatekeeper, enabling cholesterol to further penetrate the channels ensuring long channel closures. To see this figure in color, go online.

all Kir2 channels, the corresponding residues are serine and isoleucine (S94 and I172 in Kir2.2, and S95 and I171 in Kir2.1) (Figs. 6 and 7, B), which provide less steric hindrance and are less rotationally constrained. With this insight, we performed patch-clamp experiments on cholesterol-sensitive Kir2.1 channels (11). WT Kir2.1 channels were sensitive to cholesterol depletion by MbCD, which induced a relief of channel inhibition, and an increase in current density (see Fig. 9). S95H mutant Kir2.1 channels were now no longer activated following by cholesterol depletion,

Cholesterol-Binding Pocket in Kir Channels

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Slide-helix

TM1

Kir1.1 Kir2.1 Kir2.2 Kir2.3 Kir2.4 Kir2.6 Kir3.1 Kir3.2 Kir3.3 Kir3.4 Kir4.1 Kir4.2 Kir5.1 Kir6.1 Kir6.2 Kir7.1

-------------------MNASSRNVFDTLIRVLTESMFKHLRKWVVTRFFGHSRQRARLVSKDGRCNIEFGNVEAQSRFIFFVDIWTTVLDLKWRYKMTIFITAFLGSWFFFGLLWYA ------------MGSVRTNRYSIVSSEE-DGMKLATMAVANGFGNGKS-KVHTRQQCRSRFVKKDGHCNVQFINVGEK-GQRYLADIFTTCVDIRWRWMLVIFCLAFVLSWLFFGCVFWL -----------MTAASRANPYSIVSSEE-DGLHLVTMSGANGFGNGK---VHTRRRCRNRFVKKNGQCNIEFANMDEK-SQRYLADMFTTCVDIRWRYMLLIFSLAFLASWLLFGIIFWV ------------------------------------MHGHSRNGQAH----VPRRKRRNRFVKKNGQCNVYFANLSNK-SQRYMADIFTTCVDTRWRYMLMIFSAAFLVSWLFFGLLFWC ---------MGLARALRRLSGALDSGDSRAGDEEEAGPGLCRNGWAPAPVQSPVGRRRGRFVKKDGHCNVRFVNLGGQ-GARYLSDLFTTCVDVRWRWMCLLFSCSFLASWLLFGLAFWL -----------MTAASQANPYSIVSLEE-DGLHLVTMSGAKGFGNGK---VHTQHRCRNRFVKKNGQCNIAFANMDEK-SQRYLADMFTTCVDIRWRYMLLIFSLAFLASWLLFGVIFWV MSALRR---------KFGDDYQVVTTSSSGSGLQPQGPGQDPQQQLVP------KKKRQRFVDKNGRCNVQHGNLGSE-TSRYLSDLFTTLVDLKWRWNLFIFILTYTVAWLFMASMWWV MAKLTESMTNVLEGDSMDQDVESPVAIHQP-KLPKQARDDLPRHISRDR----TKRKIQRYVRKDGKCNVHHGNVR-E-TYRYLTDIFTTLVDLKWRFNLLIFVMVYTVTWLFFGMIWWL --------------------MAQENAAFSP------GQEEPPR-----------RRGRQRYVEKDGRCNVQQGNVR-E-TYRYLTDLFTTLVDLQWRLSLLFFVLAYALTWLFFGAIWWL MAGDSR--------NAMNQDMEIGVTPWDPKKIPKQARDYVPIATDRTRLLAEGKKPRQRYMEKSGKCNVHHGNVQ-E-TYRYLSDLFTTLVDLKWRFNLLVFTMVYTVTWLFFGFIWWL --------------------MTSVAKVYYSQTTQTES-----------RPLMGPGIRRRRVLTKDGRSNVRMEHIADK-RFLYLKDLWTTFIDMQWRYKLLLFSATFAGTWFLFGVVWYL ---------------------MDAIHIGMSSTPLVKH-----------TAGAGLKANRPRVMSKSGHSNVRIDKVDGI-YLLYLQDLWTTVIDMKWRYKLTLFAATFVMTWFLFGVIYYA -------------------------MSYYGSSYHIINADAKYPGYPPEHIIAEKRRARRRLLHKDGSCNVYFKHIFGE-WGSYVVDIFTTLVDTKWRHMFVIFSLSYILSWLIFGSVFWL -------------------MLARKSIIPEEYVLARIAAENLRKPRIRD------RLPKARFIAKSGACNLAHKNIR-E-QGRFLQDIFTTLVDLKWRHTLVIFTMSFLCSWLLFAIMWWL -------------------MLSRKGIIPEEYVLTRLAED-PAKPRYRA------RQRRARFVSKKGNCNVAHKNIR-E-QGRFLQDVFTTLVDLKWPHTLLIFTMSFLCSWLLFAMAWWL -----------------------MDSSNCKVIAPLLS------------------QRYRRMVTKDGHSTLQMDGAQRG--LAYLRDAWGILMDMRWRWMMLVFSASFVVHWLVFAVLWYV * : *.* ..: :. * : :* :* .* : *:.:. ::

Kir1.1 Kir2.1 Kir2.2 Kir2.3 Kir2.4 Kir2.6 Kir3.1 Kir3.2 Kir3.3 Kir3.4 Kir4.1 Kir4.2 Kir5.1 Kir6.1 Kir6.2 Kir7.1

VAYIHKDLPEFHPSANHTP---------------CVENINGLTSAFLFSLETQVTIGYGFRCVTEQCATAIFLLIFQSILGVIINSFMCGAILAKISRPKKRAKTITFSKNAVISKRGGK IALLHGDLDASK-EGK-----------------ACVSEVNSFTAAFLFSIETQTTIGYGFRCVTDECPIAVFMVVFQSIVGCIIDAFIIGAVMAKMAKPKKRNETLVFSHNAVIAMRDGK IAVAHGDLEPAEGRGRT----------------PCVMQVHGFMAAFLFSIETQTTIGYGLRCVTEECPVAVFMVVAQSIVGCIIDSFMIGAIMAKMARPKKRAQTLLFSHNAVVALRDGK IAFFHGDLEASPGVPAAGGPAAGGGGAAPVAPKPCIMHVNGFLGAFLFSVETQTTIGYGFRCVTEECPLAVIAVVVQSIVGCVIDSFMIGTIMAKMARPKKRAQTLLFSHHAVISVRDGK IASLHGDLAAPPPPAP------------------CFSHVASFLAAFLFALETQTSIGYGVRSVTEECPAAVAAVVLQCIAGCVLDAFVVGAVMAKMAKPKKRNETLVFSENAVVALRDHR IAVAHGDLEPAEGHGRT----------------PCVMQVHGFMAAFLFSIETQTTIGYGLRCVTEECLVAVFMVVAQSIVGCIIDSFMIGAIMAKMARPKKRAHTLLFSHNAVVALRDGK IAYTRGDLNK--AHVGNYTP--------------CVANVYNFPSAFLFFIETEATIGYGYRYITDKCPEGIILFLFQSILGSIVDAFLIGCMFIKMSQPKKRAETLMFSEHAVISMRDGK IAYIRGDMDH--IEDPSWTP--------------CVTNLNGFVSAFLFSIETETTIGYGYRVITDKCPEGIILLLIQSVLGSIVNAFMVGCMFVKISQPKKRAETLVFSTHAVISMRDGK IAYGRGDLEH--LEDTAWTP--------------CVNNLNGFVAAFLFSIETETTIGYGHRVITDQCPEGIVLLLLQAILGSMVNAFMVGCMFVKISQPNKRAATLVFSSHAVVSLRDGR IAYIRGDLDH--VGDQEWIP--------------CVENLSGFVSAFLFSIETETTIGYGFRVITEKCPEGIILLLVQAILGSIVNAFMVGCMFVKISQPKKRAETLMFSNNAVISMRDEK VAVAHGDLLELDPPANHTP---------------CVVQVHTLTGAFLFSLESQTTIGYGFRYISEECPLAIVLLIAQLVLTTILEIFITGTFLAKIARPKKRAETIRFSQHAVVASHNGK IAFIHGDLEPGEPISNHTP---------------CIMKVDSLTGAFLFSLESQTTIGYGVRSITEECPHAIFLLVAQLVITTLIEIFITGTFLAKIARPKKRAETIKFSHCAVITKQNGK IAFHHGDLLNDPDITP------------------CVDNVHSFTGAFLFSLETQTTIGYGYRCVTEECSVAVLMVILQSILSCIINTFIIGAALAKMATARKRAQTIRFSYFALIGMRDGK VAFAHGDIYAYMEKSGMEKSG--------LESTVCVTNVRSFTSAFLFSIEVQVTIGFGGRMMTEECPLAITVLILQNIVGLIINAVMLGCIFMKTAQAHRRAETLIFSRHAVIAVRNGK IAFAHGDLAP---SEGTAEP--------------CVTSIHSFSSAFLFSIEVQVTIGFGGRMVTEECPLAILILIVQNIVGLMINAIMLGCIFMKTAQAHRRAETLIFSKHAVIALRHGR LAEMNGDLELDHDAPPENHT-------------ICVKYITSFTAAFSFSLETQLTIGYGTMFPSGDCPSAIALLAIQMLLGLMLEAFITGAFVAKIARPKNRAFSIRFTDTAVVAHMDGK :* . *: *. : : .** * :* : :**:* : .* .: . * : ::: .: * . * : ...* :: *: *:: :

101 105 104 79 110 104 104 113 81 110 88 87 94 93 92 77

TM2 206 207 208 199 212 208 208 217 185 214 193 192 196 205 195 184

FIGURE 6 Sequence alignment of eukaryotic Kir channels. Highlighted in yellow are the conserved residues found to interact with cholesterol in Kir2.2. The black arrows indicate the sequence of the protein used as the search-space for docking calculations. The light blue arrows indicate residues H67 and L148 in Kir7.1 correspond to residues S95 and I171 in Kir2.1 channels. To see this figure in color, go online.

whereas I171L and S95H/I171L Kir2.1 currents were actually reduced by depletion of membrane cholesterol (see Fig. 9). This may be due to regulation of Kir channel currents by other membrane properties (such as membrane thickness, fluidity, lateral pressure, etc.) that are also affected by the removal of membrane cholesterol. Nevertheless, the striking loss of cholesterol inhibition in these mutants provides experimental validation of the biological relevance of the binding pocket that is predicted computationally, and implicates sterol-protein interaction at this location, which can be weakened by appropriate mutations that lead to steric hindrance of the ligand. DISCUSSION A growing number of ion channels have been shown to be regulated by specific components of the lipid membranes (8–10,12,14,15,17,21–23,25–27), and previous studies have indicated that cholesterol can inhibit both mammalian and bacterial Kir channel currents (12,21–23,25–27). We have shown that prokaryotic KirBac1.1, KirBac3.1, or human Kir2.1 are all similarly inhibited by increasing concentrations of cholesterol in otherwise POPE/POPG liposomes (25), but that enantiomeric cholesterol has no effect (25). Previous studies have shown that sterol effects on lipid membrane properties are typically not enantioselective,

with no differences on phase-transition properties (41) and lipid packing in monolayers, and on bilayer properties as measured by calorimetry, x-ray diffraction, and neutron density measurements reported between cholesterol and entcholesterol (29). As a result, the differential functional effect of cholesterol and ent-cholesterol is a useful indicator of specific sterol-protein interactions rather than indirect effects on membrane properties (28–30,41–43). Enantioselectivity of cholesterol regulation indicates that direct sterol-protein interactions are common to numerous channels and receptors (for complete list see review by Covey, 2009 (44)), including calcium-activated BK channels (45,46). Using computational approaches, we docked cholesterol to crystal structures of KirBac1.1 and Kir2.2 channels, and ruled out false-positive binding pockets by comparing these simulations to those performed with ent-cholesterol as the ligand. Using this strategy, in prokaryotic and eukaryotic Kir channels, we observed only one equivalent site in which cholesterol bound to the channels with a high frequency of poses and ent-cholesterol either did not bind or bound with significantly higher binding energy (Figs. 1 and 2). Close examination of this binding pocket suggests that steric clashes underlie failure of ent-cholesterol interaction (Fig. 3). Because the degree of channel modulation by cholesterol is similar in prokaryotic and eukaryotic Kir channels, we Biophysical Journal 107(12) 2786–2796

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FIGURE 7 Cholesterol and Kir7.1 channels. Kir7.1 channels have been previously shown to be insensitive to cholesterol. As most favorably docked in Kir2.2, cholesterol would sterically clash with H67 and L148 residues of Kir7.1 (highlighted by black circle) (A). The equivalent residues in both Kir2.1 and Kir2.2 channels are a serine and isoleucine, respectively (B), which provide sufficient space for the cholesterol tail to interact with the channel. To see this figure in color, go online.

thus speculate that modulation of both eukaryotic and prokaryotic Kirs by cholesterol uses the same conserved binding pocket, even though cholesterol is not present in prokaryotic membranes (47). Indeed, cholesterol appears Biophysical Journal 107(12) 2786–2796

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to bind in the same region of the channel, between TM1 and TM2 of a single subunit, with the hydroxyl group interacting with the slide helix (Fig. 4). PIP2 binds to, and activates, eukaryotic Kir channels at a defined site, but importantly, cholesterol docks to a unique distinct site (see Fig. 10), suggesting allosteric interactions between the two ligands, rather than competition of cholesterol for PIP2. This is consistent with previous findings that cholesterol effects on Kir2.1 current density were not correlated with current rundown induced by PIP2-depletion. Cholesterol sensitivities of Kir2.1/Kir2.3 channels were also unaffected by decreased PIP2 availability, further arguing for PIP2-independent cholesterol regulation in these channels (48). Together, these observations may provide the molecular basis by which cholesterol locks Kir channels in a prolonged closed conformation (25) by bridging the TM1, TM2, and slide helices together. Kir7.1 channel activity has previously been shown to be unaffected by cholesterol depletion (22). Therefore, to help guide mutagenesis studies, we generated a homology model of Kir7.1 channels with the assumption that relevant cholesterol binding in Kir2.2 would be absent. When we attempted to position cholesterol in the equivalent pocket in Kir7.1 to that occupied in the Kir2.2 model, steric clashes were observed between the cholesterol carbon tail and residues H67 and L148 (Fig. 7). Notably, H67 has little rotational freedom, and cannot rotate to avoid this steric clash. Kir7.1 channels are the only Kir isoforms that contain this pair of residues, with all other (except Kir6 channels) having highly conserved serines and isoleucines in these positions, respectively. With this insight, we mutated the equivalent residues in Kir2.1 channels to S95H and I171L and observed significant loss of the effects of cholesterol depletion. Thus, in the presence of cholesterol (untreated cells), channel inhibition is reduced as a result of the ligand being crowded out of the binding pocket. Notably, Kir7.1 channels were able to bind cholesterol in the same pocket with high frequency (21% of poses) during docking calculations (Fig. 8), but cholesterol bound with the carbon tail flipped outward, away from the protein and with lower predicted affinity. In reality, the difference in binding affinity is likely to be even greater, because the calculations are based on diffusion in three-dimensional space, whereas cholesterol would be expected to primarily diffuse away from the channel two-dimensionally within the membrane leaflet. A recent study also showed the sensitivity to cholesterol enrichment was altered by mutations of residues, which we identify as contributing to lining the cholesterol pocket, specifically A70V, S95A/T, and M183I mutant Kir2.1 channels, whereas F73 mutations rendered the channels nonfunctional (49) (Figs. 9 and 10) Recent studies have implicated a band of residues at the top of the cytoplasmic domain in the cholesterol regulation of Kir channels, dubbed the cholesterol sensitivity belt (48,50) (Fig. 11 A). Although these residues appear to be critical

Cholesterol-Binding Pocket in Kir Channels

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1 nA

1 nA

A

B

35

*

- MβCD (untreated) + MβCD (treated)

Slope Conductance (pS)

(25)

30 25

(19)

n.s. (12)

20

*

*

(16)

(28) (13)

15

(17)

(18)

10 5 0

WT

S95H

I171L

S95H/I171L

FIGURE 9 Electrophysiological confirmation of putative cholesterolbinding pocket. (A) Cell-attached recordings of WT, S95H, I171L, and S95H/I171L Kir2.1 channels before (black) and after (red) depletion with MbCD to deplete membranes of cholesterol. Ramps were measured between 160 to þ160 mV over 320 ms from a holding potential of 0 mV in 150 mM Kþ solution. (B) Estimates of slope-conductances determined from (A). Unlike WT Kir2.1 cholesterol does not inhibit, S95H, I171L, and S95H/I171L Kir2.1 channels, providing experimental evidence for the putative binding pocket predicted by docking calculations. To see this figure in color, go online.

FIGURE 8 Computational assessment of cholesterol binding in Kir7.1 channels. (A) Docking calculations as performed for KirBac1.1 and Kir2.2 were also performed on a homology model of Kir7.1. Cholesterol was observed to bind with high frequency (21% of poses) and low energy (DG ¼ 6.01 5 0.25 (n ¼ 38)) (KKir7.1i ¼ 41.9 5 16.7 mM) in the equivalent binding pocket, but with the carbon tail flipped outward away from the channel. This is significantly weaker (P < 1  1012) compared to cholesterol bound to Kir2.2 channels with a DG ¼ 6.53 5 0.40 kcal/ mol (n ¼ 152) resulting in at least a twofold decrease in KKir2.2i (¼ 19.8 5 13.3 mM). (B) A representative pose of cholesterol docked ˚ of cholesto Kir7.1 channels (top). The frequency of residues within 5 A terol for each pose within the cluster was assessed and is indicated as a percentage (bottom). Residues that interact with cholesterol with a frequency >45% of poses are highlighted in the structure. To see this figure in color, go online.

for coupling cholesterol binding to channel gating, they may not appear to be involved in binding cholesterol directly, based on lack of interaction between cholesterol and any of these interactions in docking calculations (50). A large transfer energy penalty (>5 kBT/lipid, dominated by the solvation energy) (51,52) would need to be paid for cholesterol to exit the lipid bilayer and enter the aqueous intracellular milieu before binding to a site in the cytoplasmic domain, if one existed. The site identified in this study is strictly located within the lipid bilayer, and closely corresponds to the preferred orientation of cholesterol within a membrane (51,52). Thus, it is likely that cholesterol binding to the site we have identified, triggers conformational changes in the cytoplasmic domain (perhaps transduced through the cholesterol sensitivity belt) that enhance channel closure. Even more recently, an attempt to identify putative cholesterol binding sites in Kir2.1 channels was performed in a similar manner to the studies performed here (49). The Kir2.1 homology model used was derived from a Biophysical Journal 107(12) 2786–2796

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FIGURE 10 Cholesterol, PIP2, and PPA. Cholesterol bound within the putative binding site we identify here does not overlap with PIP2 (A) or PPA (B) as observed in the Kir2.2 channel structures. To see this figure in color, go online.

Kir3.1-KirBac chimera (Potassium Channel Databank (KDB): H011), although docking calculations used in this study used the strictly eukaryotic Kir2.2 atomic structure (PDB: 3SPI). A comparison of the putative binding region in these structures is presented in Fig. 11 B. Notably, in the atomic structures of eukaryotic Kir channels (33,53,54), the slide helices kink outward following the second helical turn. However, in the chimeric structure (55), and other KirBac structures (32,56), the slide helix extends an additional full helical turn beyond the kink observed in eukaryotic channels. Cholesterol docked in orientations that we observed in our study would clash with the extra turn of the slide helix, and thus would not likely have been identified in that previous study. Noticeably, the cholesterol molecule in this orientation clashes with the end of the slide helix in the chimeric structure, but in the Kir2.2 structure, the slide helix kinks before this point, allowing cholesterol to dock cleanly, avoiding all steric clashes. Similar clashes Biophysical Journal 107(12) 2786–2796

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FIGURE 11 Location of residues involved in cholesterol regulation of Kir channels. (A) Cytoplasmic residues within the cholesterol sensitivity belt of Kir2.1 (highlighted in the dashed box) consist of residues D51 and H53 (cyan), E191 and V194 (blue), N216 and K219 (pink), L222 (red), and C311 (green) appear to be important for cholesterol regulation of channel gating but not through direct binding (50). Docking calculations in Kir2.2 suggest cholesterol docks within the lipid bilayer region in a pocket lined by residues A68 [70], D69 [71], M70 [I72], F71 [F73], A89 [91], F175 [174] from chain A, and M184 [183] from chain B (orange; Kir2.1 numbering in brackets) and I172 [171] (yellow). S94 (S95 in Kir2.1) (purple) is oriented away from the cholesterol pocket but, when mutated to histidine (the equivalent residue in cholesterol-insensitive Kir7.1 channels), is sterically forced to occlude the binding site. This also applied to the I171L mutation in Kir2.1 channels. (B) Cholesterol docked in the most favorable position in Kir2.2 (PDB: 3SPI; gray) compared to a Kir2.1 model derived from a Kir3.1-KirBac chimera (KDB: H011; pink). Noticeably, the cholesterol molecule in this orientation clashes with the end of the slide helix in the chimeric structure, whereas in the Kir2.2 structure the slide helix kinks before this point, allowing cholesterol to dock and avoiding all steric clashes. To see this figure in color, go online.

were avoided in our docking calculations to KirBac1.1, because the cholesterol molecule bound deeper into the cleft, enabled by a rotamerization of a phenylalanine residue in the TM1 that may act as a gatekeeper (Fig. 5, C and D).

Cholesterol-Binding Pocket in Kir Channels

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However, both our sets of docking calculations and MD simulations and those performed by Rosenhouse-Dantsker et al. (49), lead to a consensus binding region in the cytoplasmic half of the TM domains of Kir channels with the ligand interacting with residues on the slide helix, and helices of two neighboring subunits.

10. Martens, J. R., R. Navarro-Polanco, ., M. M. Tamkun. 2000. Differential targeting of Shaker-like potassium channels to lipid rafts. J. Biol. Chem. 275:7443–7446.

CONCLUSIONS

13. Ambudkar, I. S. 2004. Cellular domains that contribute to Ca2þ entry events. Sci. STKE. 2004:pe32.

We have used computational docking calculations and MD simulations to predict a putative cholesterol binding site in prokaryotic and eukaryotic Kir channels, which is confirmed experimentally in eukaryotic channels by electrophysiological methods. The data provide a structural model for cholesterol binding in Kir channels, as well as explanation both for the lack of interaction between enantiomeric cholesterol and Kir channels, and for lack of sensitivity of Kir7.1 channels to cholesterol inhibition.

14. Bowles, D. K., C. L. Heaps, ., E. M. Price. 2004. Hypercholesterolemia inhibits L-type calcium current in coronary macro-, not microcirculation. J. Appl. Physiol. 96:2240–2248.

We thank Dr. Sunjoo Lee for her assistance establishing the docking calculation methodology, and Dr. Brett Olson for kindly supplying cholesterol and ent-cholesterol structure and charge information for parameterization. Finally, we thank Dr. Rikard Blunck and members of his laboratory for the use of his electrophysiology setup. This work was funded by research grants from the National Institutes of Health (HL54171 to CGN; K99 HL112300-01 to N.D.) and by the Canadian Institute of Health Research (OCN126568 to N.D.) as well as a Grant-in-aid from the Heart & Stroke Foundation of Canada (G-130001882 to ND). Computational resources were provided by Calcul Que´bec.

REFERENCES 1. Beeler, Jr., G. W., and H. Reuter. 1970. Voltage clamp experiments on ventricular myocarial fibres. J. Physiol. 207:165–190. 2. Frindt, G., and L. G. Palmer. 1989. Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 256:F143–F151. 3. Ga¨hwiler, B. H., and D. A. Brown. 1985. GABAB-receptor-activated Kþ current in voltage-clamped CA3 pyramidal cells in hippocampal cultures. Proc. Natl. Acad. Sci. USA. 82:1558–1562. 4. Inagaki, N., Y. Tsuura, ., S. Seino. 1995. Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart. J. Biol. Chem. 270:5691–5694. 5. Takahashi, T. 1990. Inward rectification in neonatal rat spinal motoneurones. J. Physiol. 423:47–62. 6. Wang, W. H., A. Schwab, and G. Giebisch. 1990. Regulation of smallconductance Kþ channel in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 259:F494–F502. 7. Rougier, O., G. Vassort, and R. Sta¨mpfli. 1968. Voltage clamp experiments on frog atrial heart muscle fibres with the sucrose gap technique. Pflugers Arch. Gesamte Physiol. Menschen Tiere. 301:91–108. 8. Bolotina, V., V. Omelyanenko, ., P. Bregestovski. 1989. Variations of membrane cholesterol alter the kinetics of Ca2(þ)-dependent Kþ channels and membrane fluidity in vascular smooth muscle cells. Pflugers Arch. 415:262–268. 9. Heaps, C. L., D. L. Tharp, and D. K. Bowles. 2005. Hypercholesterolemia abolishes voltage-dependent Kþ channel contribution to adenosine-mediated relaxation in porcine coronary arterioles. Am. J. Physiol. Heart Circ. Physiol. 288:H568–H576.

11. Romanenko, V. G., Y. Fang, ., I. Levitan. 2004. Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels. Biophys. J. 87:3850–3861. 12. Romanenko, V. G., G. H. Rothblat, and I. Levitan. 2002. Modulation of endothelial inward-rectifier Kþ current by optical isomers of cholesterol. Biophys. J. 83:3211–3222.

15. Lockwich, T. P., X. Liu, ., I. S. Ambudkar. 2000. Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains. J. Biol. Chem. 275:11934–11942. 16. Lundbaek, J. A., P. Birn, ., O. S. Andersen. 1996. Membrane stiffness and channel function. Biochemistry. 35:3825–3830. 17. Toselli, M., G. Biella, ., M. Parenti. 2005. Caveolin-1 expression and membrane cholesterol content modulate N-type calcium channel activity in NG108-15 cells. Biophys. J. 89:2443–2457. 18. Lundbaek, J. A., P. Birn, ., O. S. Andersen. 2004. Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol. J. Gen. Physiol. 123:599–621. 19. Wu, C. C., M. J. Su, ., Y. T. Lee. 1995. The effect of hypercholesterolemia on the sodium inward currents in cardiac myocyte. J. Mol. Cell. Cardiol. 27:1263–1269. 20. Levitan, I., A. E. Christian, ., G. H. Rothblat. 2000. Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells. J. Gen. Physiol. 115:405–416. 21. Romanenko, V. G., G. H. Rothblat, and I. Levitan. 2004. Sensitivity of volume-regulated anion current to cholesterol structural analogues. J. Gen. Physiol. 123:77–87. 22. Rosenhouse-Dantsker, A., E. Leal-Pinto, ., I. Levitan. 2010. Comparative analysis of cholesterol sensitivity of Kir channels: role of the CD loop. Channels (Austin). 4:63–66. 23. Tikku, S., Y. Epshtein, ., I. Levitan. 2007. Relationship between Kir2.1/Kir2.3 activity and their distributions between cholesterol-rich and cholesterol-poor membrane domains. Am. J. Physiol. Cell Physiol. 293:C440–C450. 24. Rosenhouse-Dantsker, A., S. Noskov, ., I. Levitan. 2012. Distant cytosolic residues mediate a two-way molecular switch that controls the modulation of inwardly rectifying potassium (Kir) channels by cholesterol and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). J. Biol. Chem. 287:40266–40278. 25. D’Avanzo, N., K. Hyrc, ., C. G. Nichols. 2011. Enantioselective protein-sterol interactions mediate regulation of both prokaryotic and eukaryotic inward rectifier Kþ channels by cholesterol. PLoS ONE. 6:e19393. 26. Singh, D. K., A. Rosenhouse-Dantsker, ., I. Levitan. 2009. Direct regulation of prokaryotic Kir channel by cholesterol. J. Biol. Chem. 284:30727–30736. 27. Singh, D. K., T. P. Shentu, ., I. Levitan. 2011. Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein. Biochim. Biophys. Acta. 1808:2527–2533. 28. Alakoskela, J. M., K. Sabatini, ., P. K. Kinnunen. 2008. Enantiospecific interactions between cholesterol and phospholipids. Langmuir. 24:830–836. 29. Mannock, D. A., T. J. McIntosh, ., R. N. McElhaney. 2003. Effects of natural and enantiomeric cholesterol on the thermotropic phase behavior and structure of egg sphingomyelin bilayer membranes. Biophys. J. 84:1038–1046. 30. Westover, E. J., and D. F. Covey. 2003. First synthesis of ent-desmosterol and its conversion to ent-deuterocholesterol. Steroids. 68:159–166. Biophysical Journal 107(12) 2786–2796

2796 31. Clarke, O. B., A. T. Caputo, ., J. M. Gulbis. 2010. Domain reorientation and rotation of an intracellular assembly regulate conduction in Kir potassium channels. Cell. 141:1018–1029. 32. Kuo, A., J. M. Gulbis, ., D. A. Doyle. 2003. Crystal structure of the potassium channel KirBac1.1 in the closed state. Science. 300:1922– 1926. 33. Hansen, S. B., X. Tao, and R. MacKinnon. 2011. Structural basis of PIP2 activation of the classical inward rectifier Kþ channel Kir2.2. Nature. 477:495–498. 34. Olsen, B. N., P. H. Schlesinger, and N. A. Baker. 2009. Perturbations of membrane structure by cholesterol and cholesterol derivatives are determined by sterol orientation. J. Am. Chem. Soc. 131:4854–4865. 35. Larkin, M. A., G. Blackshields, ., D. G. Higgins. 2007. Clustal W and Clustal X version 2.0. Bioinformatics. 23:2947–2948. 36. Abagyan, R., W. H. Lee, ., B. D. Marsden. 2006. Disseminating structural genomics data to the public: from a data dump to an animated story. Trends Biochem. Sci. 31:76–78.

Fu¨rst et al. 44. Covey, D. F. 2009. ent-Steroids: novel tools for studies of signaling pathways. Steroids. 74:577–585. 45. Bukiya, A. N., J. D. Belani, ., A. M. Dopico. 2011. Specificity of cholesterol and analogs to modulate BK channels points to direct sterol-channel protein interactions. J. Gen. Physiol. 137:93–110. 46. Yuan, C., M. Chen, ., S. N. Treistman. 2011. Cholesterol tuning of BK ethanol response is enantioselective, and is a function of accompanying lipids. PLoS ONE. 6:e27572. 47. Salton, M. R. 1971. The bacterial membrane. In Biomembranes. L. A. Manson, editor. Plenum Press, New York, pp. 1–65. 48. Epshtein, Y., A. P. Chopra, ., I. Levitan. 2009. Identification of a Cterminus domain critical for the sensitivity of Kir2.1 to cholesterol. Proc. Natl. Acad. Sci. USA. 106:8055–8060. 49. Rosenhouse-Dantsker, A., S. Noskov, ., I. Levitan. 2013. Identification of novel cholesterol-binding regions in Kir2 channels. J. Biol. Chem. 288:31154–31164.

37. Sanner, M. F. 1999. Python: a programming language for software integration and development. J. Mol. Graph. Model. 17:57–61.

50. Rosenhouse-Dantsker, A., D. E. Logothetis, and I. Levitan. 2011. Cholesterol sensitivity of KIR2.1 is controlled by a belt of residues around the cytosolic pore. Biophys. J. 100:381–389.

38. Jo, S., T. Kim, and W. Im. 2007. Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PLoS ONE. 2:e880.

51. Kessel, A., N. Ben-Tal, and S. May. 2001. Interactions of cholesterol with lipid bilayers: the preferred configuration and fluctuations. Biophys. J. 81:643–658.

39. Jo, S., T. Kim, ., W. Im. 2008. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29:1859–1865.

52. Oren, I., S. J. Fleishman, ., N. Ben-Tal. 2004. Free diffusion of steroid hormones across biomembranes: a simplex search with implicit solvent model calculations. Biophys. J. 87:768–779. 53. Whorton, M. R., and R. MacKinnon. 2011. Crystal structure of the mammalian GIRK2 Kþ channel and gating regulation by G proteins, PIP2, and sodium. Cell. 147:199–208.

40. Phillips, J. C., R. Braun, ., K. Schulten. 2005. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26:1781–1802. 41. Epand, R. M., S. D. Rychnovsky, ., R. F. Epand. 2005. Role of chirality in peptide-induced formation of cholesterol-rich domains. Biochem. J. 390:541–548. 42. Liu, J., C. C. Chang, ., T. Y. Chang. 2005. Investigating the allosterism of acyl-CoA:cholesterol acyltransferase (ACAT) by using various sterols: in vitro and intact cell studies. Biochem. J. 391:389–397.

54. Whorton, M. R., and R. MacKinnon. 2013. X-ray structure of the mammalian GIRK2-bg G-protein complex. Nature. 498:190–197.

43. Wang, J., F. Sun, ., X. S. Xie. 2006. Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution. J. Biol. Chem. 281:27894– 27904.

56. Bavro, V. N., R. De Zorzi, ., S. J. Tucker. 2012. Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating. Nat. Struct. Mol. Biol. 19:158–163.

Biophysical Journal 107(12) 2786–2796

55. Nishida, M., M. Cadene, ., R. MacKinnon. 2007. Crystal structure of a Kir3.1-prokaryotic Kir channel chimera. EMBO J. 26:4005–4015.