Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1*

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K+ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K+ channel ligands can expand in the subunit interface whose residues are less conserved between K+ channels and hence may be targets for design of highly desirable subtype-specific K+ channel drugs.

sequences. Each P-loop contains an extracellular S5-P linker, the pore helix (P-helix), the ascending limb with the signature sequence TVGYG, and the extracellular linker P-S6 (1).
Potassium channels are blocked by drugs of dramatically different chemical structures (2,3). Pore-lining residues in S6 segments and P-loop turns affect binding of open channel blockers (4). In the experimentally determined (5-7) and predicted (8 -11) structures of K ϩ channels with blockers, the latter occupy the central cavity.
In x-ray structures of K ϩ channels, four radial niches are seen between neighboring S6 segments and P-helices. Mutations in the niches affect binding of some K ϩ channel blockers (9,12). The niche-lining residues are less conserved than the pore-lining residues and therefore may be targets for design of subtypespecific ligands of K ϩ channels (2).
An important step toward this goal is to understand how currently available Kv2.1-selective ligands block the channel. Structurally different antiarrhythmics propafenone (PROP) 4 and flecainide (FLEC) block Kv2.1 more potently than other Kv channels (12,27). Earlier systematic substitution of segments in PROP-sensitive channel Kv2.1 with those from PROP low-sensitive channel Kv1.2 allowed to identify five loci in the P-helix and S6, which are responsible for Kv2.1 selectivity to PROP (12). In the present study, we found that substitution of some of the PROP-sensing loci in Kv2.1 also decreased FLEC potency. To rationalize this observation, we created a Kv1.2-based homology model of Kv2.1 and used multiple Monte Carlo-energy minimizations (28) to search for energetically preferable binding modes. Computations predict that a large part of the FLEC molecule fits the niche between P-helix and S6, where trifluoromethyl groups bind to loci TIT p46 (see Table 1 for residue labels) and V i17 , whereas the ammonium group exposes to the central cavity. A large part of the PROP molecule also binds in the niche, where it interacts with the TIT p46 locus and approaches V i17 , whereas the aminopropyl group protrudes in the central cavity.
Our study, for the first time, identifies FLEC-sensing residues in Kv2.1, provides structural models of PROP and FLEC in Kv2.1, defines loci with direct and indirect effects on FLEC and PROP potencies, and highlights importance of the niches between P-helices and inner helices as targets for subtype-specific K ϩ channel blockers.

MATERIALS AND METHODS
In Vitro Mutagenesis and RNA Synthesis-The cDNAs for chimeras of rat Kv1.2 (GenBank TM accession no. X16003) and human Kv2.1 (GenBank TM accession no. X68302) were obtained using an overlap PCR and were cloned into the pGEM vector (29). The chimera details are given in the supplemental information. PCR-amplified DNA sequences were verified by using the BigDye terminator cycle sequencing kit (PerkinElmer Life Sciences). The sequence reactions were analyzed on an ABI 377 or Prism 310 automated sequencer (PerkinElmer Life Sciences). The cDNA encoding rat Kv1.2 and human Kv2.1 and their chimeras were transcribed to cRNA using a commercial kit (mMESSAGE mMACHINE, Ambion, Austin, TX) and T7 RNA polymerase. Denaturing agarose gel electrophoresis was used to check the quality of cRNA product of each reaction and to quantify the yield.
Preparation of Oocytes-South African clawed frogs (Xenopus laevis) were anesthetized in ethyl m-aminobenzoate (Sandoz, Basel, Switzerland), and small sections of the ovary were removed surgically. Oocytes were injected with 0.1 or 1.0 ng of cRNA in 50 nl of distilled water and were maintained under tissue culture conditions at 20°C until used for experiments. The tissue culture solution was a modified Barth medium: 88 mmol/liter NaCl, 1 mmol/liter KCl, 1.5 mmol/liter CaCl 2 , 2.4 mmol/liter NaHCO 3 , 0.8 mmol/liter MgSO 4 , 5 mmol/liter HEPES, pH 7.4, which was supplemented with penicillin (100 international units/ml) and streptomycin (100 g/ml).
Electrophysiological Techniques-Oocytes with follicular tissues were investigated with the two-electrode voltage clamp technique. Microelectrodes were made from borosilicate glass and had resistances of 0.5-1 megohm for the current electrodes and 1-2 megohm for the potential electrodes when filled with 3 mol/liter KCl. The holding potential was Ϫ80 mV, and command potentials were applied up to a potential of ϩ60 mV. The control bath fluid was a Ringer solution consisting of the following: 115 mmol/liter NaCl, 2 mmol/liter KCl, 1.8 mmol/liter CaCl 2 , 10 mmol/liter HEPES, pH 7.2. FLEC (acetate salt; up to 1000 mol/liter, Meda Pharma), PROP (chloride salt; Sigma), and the R-and S-enantiomers of flecainide hydrochloride and propafenone (Dr. Margarete Fischer-Bosch, Institute for Clinical Pharmacology) was added to the bath solution and applied at least 30 s before eliciting currents. Solutions were applied with a concentration clamp technique (30), allowing an exchange of Ͼ90% of the extracellular solution within Ͻ10 ms. All experiments were performed at days 3 and 4 after injection of cRNA and were carried out at room temperature (22 Ϯ 1°C).
Data Acquisition and Analysis-The K ϩ currents obtained in two-electrode recordings were low pass-filtered at 1 kHz and transferred to a computer (pClamp program, Axon Instru-ments). The amplitudes of the total outward currents were corrected for leakage. Leakage currents and capacitive transients were subtracted online using a p/Ϫ4 pulse protocol.
The K ϩ current amplitudes were measured at the end of 500-ms depolarizing voltage steps. Conductance-voltage relations were obtained by normalizing the conductance data to the maximal value under control conditions and by fitting the data to the Boltzmann equation where y is the normalized conductance, G max is the normalized maximal conductance, V1 ⁄ 2 is the potential of the half-maximal conductance, V is the voltage, and b is the slope factor. Concentration-response curves were determined by fitting the mean current values at different FLEC concentrations to the Langmuir equation y ϭ (K D /c) n /(1 ϩ (K D /c) n )), where y is the fraction of control current, K D is the half-blocking concentration, c is the FLEC concentration, and n is the Hill coefficient.
The voltage dependence of block was determined using the K D values, which were obtained from the fractional current represents the half-blocking concentration at the reference potential of 0 mV, V is the membrane potential, and z, F, R, and T have their usual meanings.
The measured values are given as mean Ϯ S.E. Statistical significance was tested using a t test or a Mann-Whitney rank sum test. Values of p Յ 0.01 were taken as statistically significant. Curve fitting and all statistical procedures were performed using the program SigmaPlot (Jandel Scientific, Erkrath, Germany).
Molecular Modeling-The homology model of Kv2.1 was based on a Kv1.2 x-ray structure, Protein Data Bank code 2A79 (31). Energetically optimal drug-channel complexes were searched by the Monte Carlo-energy minimization method (32) from multiple starting points using the ZMM program as described elsewhere (28). The starting positions and orientations of the drugs were seeded in the area covering all drugsensing loci, except that in the P-S6 linker, which is not reachable from the inner pore. An AMBER force field (33) with a cut-off distance of 8 Å was used. The hydration energy was computed using the implicit solvent method (34). All ionizable residues were kept in a neutral form (35). Atomic charges of FLEC and PROP were calculated by AM1 method of MOPAC (36). Four potassium binding sites in the outer pore, T1-T4, were loaded as follows: sites T1 and T3 with K ϩ ions and sites T2 and T4 with explicit water molecules. Residues are designated (see Table 1) using a scheme universal for P-loop channels (37). The voltage dependence of block was calculated by the fraction of control current with FLEC in the potential range from Ϫ30 to ϩ60 mV (Fig. 1C). The FLEC-induced block increased with positive going potentials as indicated by a steady decrease of the half-blocking concentrations (K D ). The fractional electrical distance (␦), i.e. the fraction of the transmembrane electrical field sensed by the drug at the binding site referenced to the intracellular side of the channel, was calculated at potentials from ϩ40 to ϩ60 mV. In this potential range, the open probability was roughly maximal (change of maximal conductance Ͻ 9%; n ϭ 8; Fig. 1C, hatched line), and the endogenous currents of the oocytes were small (Ͻ0.8 A up to ϩ80 mV in waterinjected oocytes; n ϭ 3; data not shown). With a pK a value of 9.3 for FLEC acetate and an intracellular pH value of 8.2 in oocytes (38), Ͼ90% of the intracellular FLEC should be positively charged. Calculating ␦ with the measured K D at the reference potential of 0 mV (K D 0 ϭ 263 mol/liter) and with the estimated charge of FLEC (z ϭ 0.9) yielded ␦ 1 ϭ 0.17 Ϯ 0.01 ( Fig. 1C; n ϭ 6). At the potential of 0 mV, however, the open probability was ϳ50% of the maximal value, suggesting that the theoretical K D for maximal open probability could be smaller than the computed one. The values were therefore refitted with the assumption of an unknown K D 0 . The fit revealed an electrical distance ␦ 2 ϭ 0.10 Ϯ 0.03 and a K D 0 of 200 Ϯ 29 mol/liter (Fig. 1C). Thus, the values of electrical distance are similar to those of PROP (␦ 1 ϭ 0.16 and ␦ 2 ϭ 0.14, (12)) and of other substances found to block ion channels at intracellular parts of the channel molecule (␦ ϭ 0.16 for tetraethylammonium (39) and bupivacaine (40)).

Block of
Effects of FLEC on Kv2.1/1.2 Chimeras-Kv1.2 channels were blocked less by FLEC than Kv2.1 channels. Thus, Kv1.2 currents showed a reduction of G max in the presence of 200 mol/ liter FLEC by 12 Ϯ 2% in comparison to 55 Ϯ 3% for Kv2.1 currents (Fig. 2). We took advantage of this different sensitivity and used a systematic set of Kv2.1/Kv1.2 chimeras to screen the channel domains for FLEC binding sites. We tested for insensitivity of the Kv2.1 channel to also discover a possible composite site of action.
In the first step, we replaced the Kv2.1 N and C termini, every segment (S1, S2, S3, S4, S5 and S6) and every linker (LS1/S2, LS2/S3, LS3/S4, LS4/S5, and LS5/S6 with and without the P-loop) by the respective parts of the Kv1.2 channel (Fig. 2). Original current recordings of the wild-type channels and the chimeras of the pore region under control conditions and with 200 mol/liter FLEC ( Fig. 2A) show that the current reduction by FLEC remains similar to Kv2.1 wild-type for the Kv2.1 chimera S5 and LP, whereas the current reduction by FLEC was strongly reduced in the chimera LS5/S6 and S6. The reduction of G max (Fig. 2B) in the chimeras substantially decreased reaching the Kv1.2 wild-type level only for the chimeras with the exchanged S5-S6 linker and the exchanged S6, whereas the exchange of the other extracellular linkers and segments did not cause a significant reduction of the block (Fig. 2B, asterisks indicate no statistical significant difference to Kv1.2 wild-type, n ϭ 5-12). These results suggest a high affinity binding site for FLEC in the pore region. This is completely in line with the findings for PROP (12), but in contrast to PROP exchanges of the N and C termini and the intracellular linker LS4/S5 did not cause statistically significant current reductions by FLEC.
In the second step, Kv2.1 mutants with exchanged residues in the P-loop and S6 segment (Table 1) were tested for sensitivity to FLEC (Fig. 3). Original recordings of some mutants are shown in Fig. 3A. Within the P-region of Kv2.1, exchange TIT p46 3 VVS and especially exchange IY p56 3 MV strongly reduced the sensitivity of the channel to FLEC, whereas the exchange AS p39 3 DA had no statistically significant effect (Fig. 3B, FLEC). Thus, the amino acid exchanges that decreased the PROP potency (12, Fig. 3B, PROP) also decrease the FLEC potency, despite the fact that quantitative effects of some exchanges are different. Exchanges TIT p46 3 VVS and IY p56 3 MV had the largest impact on the potencies of PROP and FLEC, respectively (Fig. 3B).
In the last step of this mutational approach, we replaced Kv2.1 residues in S6 (see Table 1) by those of Kv1.2 (Fig. 3). The exchange G i8 3 S and C i11 3 A in the outer part of S6 and SE i31 3 NY in the inner part of S6 did not reduce the sensitivity to FLEC. In contrast, the exchanges IPIIVN i27 3 VPVIVS and especially V i17 3 T significantly reduced the FLEC potency. The mutation of V i17 reduced FLEC potency to that in the wild-type Kv1.2 (Fig. 3B, FLEC). Thus, as for PROP, effects of mutations were found in S6, but in contrast to PROP (Fig. 3B, PROP), these were more focused on the middle of S6.
Comparison of PROP and FLEC Sites of Action-Because the effects of FLEC at Kv2.1 were similar to those described for PROP, we explored possible overlap of the receptors by measuring dose response relations of FLEC at ϩ60 mV under control conditions and in the presence of 100 mol/liter PROP shown to block around half of the maximum conductance (12). The block by PROP, however, also had a slow component with a time course Ͼ1 s (12). Therefore, even without application of FLEC, a small decrease of K ϩ currents was found with each voltage step in the presence of the PROP. To compensate for this effect, the PROP-induced reduction was estimated by fitting five voltage steps (preceding FLEC applications) to bi-exponential functions and by subtracting the extrapolated current decrease from the control current values. The experiments revealed a K D of 310 Ϯ 50 mol/liter FLEC for the reduction of Kv2.1 K ϩ currents at ϩ60 mV (Fig. 4, n ϭ 6). In the presence of PROP, K D increased to 1360 Ϯ 230 mol/liter FLEC (Fig. 4, n ϭ 7). The differences were statistically significant with p ϭ 0.002. Furthermore, the Hill coefficient, which indicates the number of FLEC molecules needed for block, decreased from 1.02 Ϯ 0.05 under control conditions to 0.75 Ϯ 0.13 in the presence of PROP. The increase of K D and the reduction of the Hill coefficient suggest that FLEC and PROP compete for the same or overlapping binding sites.
Mapping the FLEC Receptor-The current study shows that FLEC is an open channel blocker that enters the open pore from the cytoplasm. Locus IY p56 at the P-S6 linker is unlikely to directly interact with FLEC. Indeed, mutations at the linker are known to affect slow inactivation kinetics, e.g. (9). Other loci would be reachable by FLEC from the inner pore (Fig. 5A). An  Residue labels include a prefix (p for P-loop, i for the inner helix) and a relative number in the segment (4,40). OCTOBER 29, 2010 • VOLUME 285 • NUMBER 44

JOURNAL OF BIOLOGICAL CHEMISTRY 33901
unbiased multi-Monte-Carlo energy minimization search predicted low energy complexes with the FLEC ammonium group at the focus of P-helices (Fig. 5, A-C). In this respect, the complex resembles co-crystals of KcsA with tetrabutylammonium (5, 7). Bis-(trifluoroethoxy)-phenyl moiety filled a trifurcating niche between the P-helix and two S6 segments (Fig. 5B). Group m-CF 3 occupied a cavity in the niche, which is lined by V i17 , I i18 , and two flanking leucine residues from the S5 motif LILFL. Another cavity was occupied by group o-CF 3 , which approached the methyl group of T p46 (Fig. 5C). These interactions are consistent with the lipophilic properties of group CF 3 (41). Residues M p47 , T p48 , T p49 , V i15 , and I i18 , which are common between Kv1.2 and Kv2.1, also stabilize FLEC binding. In particular, T p49 donated an H-bond to the FLEC ether oxygen (Fig. 5C). Thus, multi-Monte-Carlo energy minimization docking predicted a complex, in which two of the four FLEC-sensing loci directly interact with the ligand and all functional groups of FLEC contribute to the ligand binding.
Mapping the PROP Receptor-High flexibility of PROP, which contains 12 rotatable bonds, decreases chances to find the global minimum in an unbiased search. Exchange TIT p46 3 VVS largely decreased the PROP potency (Fig. 3B) suggesting that T p44 likely forms an H-bond with PROP. Therefore, we used distance constrains to near different polar groups of PROP and T p44 and Monte Carlo-minimized corresponding complexes with and then without the constraints. The search yielded a low energy complex in which the carbonyl group of PROP accepted an H-bond from T p44 (Fig. 5D), and the hydroxy group donated an H-bond to the side chain of T p48 , whereas the ammonium group donated an H-bond to the backbone carbonyl of T p48 . The latter is at the P-loop turn and does not accept an H-bond from the P-helix backbone and hence is a particularly attractive site for the ligand H-bond donor. A disubstituted phenyl ring bound between I i18 and I i12 in neighboring subunits and approached V i17 as close as 5 Å. Another phenyl ring of the ligand bound between methyl group of T p48 and G i8 in the neighboring subunit.
Models in Fig. 5 show Kv2.1 with (R)-PROP and (S)-FLEC. We also docked (S)-PROP and (R)-FLEC. For each pair of enantiomers, the difference of ligand-binding energies calculated using solvent exposure-and distance-dependent dielectric function (42) is less than 2 kcal/mol. Because our computational methodology does not take into consideration the librational entropy and some other contributions to the free energy, the predicted binding energies are not expected to be precise enough to correlate them with statistically insignificant differences in the potency of enantiomers. However, superpositions of the channel-bound enantiomers (supplemental Fig. S1) show that the same residues of the channel interact with the same moieties of enantiomers, whereas positions, orientations, and conformations of the enantiomers are similar. For example, the ammonium groups of the FLEC enantiomers are at the focus of P-helices, close to the inner pore cavity center, whereas methylene groups of the piperidine ring fill the cavity below its center (supplemental Fig. S1A). The ammonium groups of the PROP enantiomers donate H-bonds to the backbone carbonyl of T p48 , whereas hydroxy groups of T p44 and T p48 are engaged in H-bonding with the carbonyl and hydroxy groups, respectively (supplemental Fig. S1B).
Testing the Modeling Predictions-As a test for the proposed ligand-binding modes in Kv2.1, we have transferred into Kv1.2 the residues, which have been found in our model to be critical for the FLEC and PROP binding in Kv2.1 (Fig. 3). Original tracings of the Kv2.1 mutants and the corresponding Kv1.2 mutants under control conditions and with FLEC are shown in Fig. 3A.  In complete agreement with the model, the current decrease by FLEC in Kv2.1 wild-type (arrows in Fig. 3A), which was strongly reduced by mutations TIT p46 3 VVS and especially V i17 3 T, was partly restores in Kv1.2 mutant VVS p46 3 TIT and almost completely restored in the T i17 3 V mutant (Fig. 3A). As far as G max is concerned, replacement of the Kv1.2 motif VVS p46 with motif TIT increased ϳ2-fold potency of both FLEC and PROP (Fig. 3B). A 4-fold increase of Kv1.2 sensitivity to both blockers was observed upon the point mutation T i17 3 V. Furthermore, the FLEC effect on the Kv1.2 T i17 3 V mutant showed no statistically significant difference to the FLEC effect in the Kv2.1 wildtype. We further transferred to Kv1.2 two components of the FLEC and PROP receptors in Kv2.1 in combination, TIT p46 and V i17 (Fig. 3B). The resulting chimera was ϳ3-fold more sensitive to FLEC and PROP than the wildtype Kv1.2. Thus, for unknown reasons the combined substitutions were not more effective than the single ones.
Finally, we transferred to Kv1.2 residues IY p56 , which do not interact with PROP and FLEC in our model, but had shown strong effects in the electrophysiological experiments. In line with our conclusions that FLEC and PROP do not bind at the extracellular mouth of the channel, the mutation did not change sensitivity of Kv1.2 to FLEC and PROP (Fig. 3B).

DISCUSSION
Voltage-gated K ϩ channels in general and Kv2.1 channels in particular are important drug targets. Although x-ray structures of several K ϩ channels in the open and closed states are available, structure-based design of subtype-specific K ϩ channel drugs remains a challenging problem. One of the reasons is incomplete knowledge of the drugs binding sites and binding modes. Here, we sought to explore structural determinants of selectivity for structurally different antiarrhythmics, PROP and FLEC, toward Kv2.1 versus other Kv channels. These drugs exhibit just a moderate Kv2.1-blocking potency, but this potency is higher than on other Kv channels (27).
Our electrophysiological data suggest that FLEC decreases the Kv2.1 currents by an open channel block from the cytoplasm. Thus, only a slight block of the current at the beginning of the voltage pulse was found, and the block increased with time. The current increase with FLEC was faster than under control conditions. Furthermore, the block increased with positive going potentials suggesting its correlation with the open probability. The fractional electrical distance (␦) of FLEC was in the range 0.10 -0.17, indicating that FLEC acts at the intracellular side of the channel. Several substances known to block the inner pore of K ϩ channel have similar range of ␦ values. These are internally applied tetraethylammonium with ␦ ϭ 0. 16 (39), quinidine with ␦ ϭ 0.19 (43), bupivacaine with ␦ ϭ 0.16 (44), and PROP with ␦ ϭ 0.14 -0.16 (12). All these data support our conclusion that the mechanism of Kv2.1 block by FLEC is similar to that by PROP (12).
Numerous studies indicate that blockers of the open P-loop channels bind in the inner cavity to the pore-facing residues in positions i15, i18, and i22 (37). Residues T p49 at the top of the cavity also contribute to binding of Kv blockers (45). Our present and previous (12) studies identified new loci in Kv2.1, whose exchange with corresponding residues in Kv1.2 diminished the blocking potency of FLEC and PROP. Three of the loci do not face the inner pore and two of them occur deep in the niches. Despite PROP and FLEC have dramatically different chemical structures, our results suggest that both drugs can bind in the niche and expose their hydrophobic ammonium groups to the inner pore to block the current (Fig. 5).
The ammonium group is considered traditionally as a fingerprint of hydrophobic cations like tetrabutylammonium. The ammonium nitrogen of FLEC occurs at the focus of P-helices (Fig. 5A). This position is slightly below the level where the nitrogen atom of tetrabutylammonium is seen in co-crystals of KcsA with tetrabutylammonium (5, 7). The NH 2 ϩ group of PROP, which forms an H-bond with T p48 , is ϳ4 Å away from the pore axis. At this distance, it would still repel permeating K ϩ ions electrostatically. In addition, the hydrophobic methyl group of PROP riches the pore axis below the selectivity filter, approximately at the level T5, where a K ϩ ion binds in the For clarity, one subunit is shown by green C ␣ tracing and S5 helices are not shown. K ϩ ions and water molecules in the selectivity filter region are spacefilled. Ligands (sticks with red oxygens, blue nitrogens, dark gray carbons in FLEC, and yellow carbons in PROP) are superimposed to indicate overlapping binding sites. Channel segments whose substitutions substantially affect action of FLEC and/or PROP (Table 1 and Fig. 3) are highlighted by red. Note that segment TIT p46 and residue V i17 are close to the ligands, whereas other segments are far from the ligands (see text for further explanations). B, close-up view at FLEC in Kv2.1. The piperidine ring is in the central cavity, and the aromatic ring is in the niche between the neighboring subunits, whose surfaces are colored blue and orange. A potassium ion and two water molecules in the selectivity filter are space-filled. C and D, close-up view at FLEC and PROP, respectively, in the channel. The ligands are shown by thick sticks with dark gray carbons, red oxygens, blue nitrogens, white polar hydrogens, and cyan fluorine atoms. Channel residues, whose direct interactions with the ligands are proposed in this study, are shown by thin sticks, and their carbons atoms are colored as respective backbones. Note that FLEC interacts with the proposed residues, which are located in the neighboring subunits (gray carbons of V i17 versus green carbons of T p46 and T p49 ), whereas PROP interacts with T p44 , T p48 , and V i17 in the same subunit (gray carbons). OCTOBER 29, 2010 • VOLUME 285 • NUMBER 44 central cavity of KcsA (Fig. 5, A and D). At this position, the methyl group would block the ion permeation by interfering with the hydration shell of a K ϩ ion approaching the selectivity filter.

Drugs Binding in Subunit Interface of Kv2.1 Channel
Although FLEC and PROP share ligand-sensing loci, quantitative effects of the exchange of loci on drug potency are different. Thus, exchange TIT p46 3 VVS decreased the PROP potency 30-fold, but the FLEC potency only 2-fold (Fig. 3B). This is consistent with our models where PROP accepts a strong H-bond from T p44 that is lost in the mutant, whereas FLEC accepts a weak H-bond from T p46 that would retain in the mutant. Diminished hydrophobicity of S p46 versus T p46 can account for the lower potency of FLEC in Kv1.2. Another mutation, V i17 3 T, diminished potency of FLEC almost 8-fold, but potency of PROP only 2-fold. In view of our models, the hydrophobic V i17 in Kv2.1 provides a favorable environment for the lipophilic CF 3 group of FLEC, but this environment is lost in mutant V i17 3 T. PROP potency was less sensitive to the mutation because the disubstituted phenyl ring approached V i17 but did not make direct contacts with it. Replacing locus IPIIVN i27 decreased FLEC potency by 13% but decreased PROP potency by 30% (Fig. 3B). Although I i22 lines the access path for both drugs to the pore, in our models, it interacts with PROP but not FLEC.
Exchange IY p56 3 MV in the extracellular side of P-loop had a similar effect on the potency of both drugs. This locus is far from the drugs in our models (Fig. 5A), implying that the mutation allosterically affects the potency of the drugs. Indeed, mutations in the P-S6 linker are known to affect kinetics of C-type inactivation in Kv channels (46). The similar effect of the IY p56 3 MV exchange on the potency of both drugs (Fig. 3B) also support an allosteric mechanism behind the observed effect of this exchange on the drugs potency.
Thus, mutational, electrophysiological, and modeling data of our study indicate a common binding site for FLEC and PROP in the niche between P-helix and the inner helix S6. From this niche, the drugs expose their ammonium groups in the inner pore to block the current. The interface between pore-forming subunits was proposed as the binding site of some ligands of Ca 2ϩ channels (47,48). Our present study provides evidence that some open K ϩ channel blockers can also bind mainly in the interface of the pore-forming subunits. This interface may be a new target for design of subtype-specific K ϩ channel drugs, demand for which is high (2).