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CELLULAR AND MOLECULAR
Nora Eccles Harrison Cardiovascular Research and Training Institute and Department of Physiology, University of Utah, Salt Lake City, Utah (L.C., K.K., M.C.S.); and Bristol-Myers Squibb, Computer Assisted Drug Design, Wallingford, Connecticut (R.R.)
Received for publication
February 15, 2007
Accepted
March 29, 2007.
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Abstract |
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100-fold less sensitive to block by ZD7288. Substitution of Ile432 with more hydrophobic residues (Phe, Leu, or Val) caused only modest shifts in the IC50 for the drug. HCN1 channels have a Val (Val390) in the equivalent position of Ile432 and are less sensitive to block by ZD7288. Accordingly, mutation of this Val390 to Ile in HCN1 increased the sensitivity of these channels to drug block. Mutation of Ala425 and Ile432 also attenuated the block of HCN2 by the more potent blocker cilobradine. An HCN2 homology model based on the bacterial KcsA K+ channel predicts that the phenyl ring of ZD7288 occupies a hydrophobic cavity formed by Ala425 and Ile432 and that the charged ring aligns with the axis of the inner pore closely corresponding to the localization of K+ ions observed in the KcsA crystal structure.
) and synaptic integration and network rhythmicity of central neurons (Biel et al., 1999; Santoro and Tibbs, 1999; Robinson and Siegelbaum, 2003). The pacemaker current is abbreviated Ih ("hyperpolarization"-activated) in neurons and If ("funny") in cardiac cells. The molecular identity of this current in mammals was first identified by Santoro et al. (1997), and the channels are now known to be formed by homomeric or heteromultimeric coassembly of four hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel subunits (Ludwig et al., 1998). All four known mammalian HCN channel subunits (HCN1–4) are expressed in the brain (Santoro et al., 1997, 1998; Ludwig et al., 1998). Northern blot and polymerase chain reaction analyses indicate HCN2 and HCN4 are expressed throughout the human heart (Ludwig et al., 1999). If channels in the sinoatrial node are composed mainly of HCN4 subunits, with lesser roles postulated for HCN1 and HCN2 (Baruscotti et al., 2005). HCN2 channels have a more negative voltage range for activation than HCN1 or HCN4 channels (Accili et al., 2002) and, in this respect, resemble If recorded from Purkinje fibers and ventricular myocytes. Thus, expression of the different isoforms of HCN determines the voltage dependence, kinetics, and magnitude of If in various regions of the heart.
Mutations in the HCN4 gene causes sick sinus syndrome, characterized by a very slow intrinsic heart rate (Schulze-Bahr et al., 2003; Ueda et al., 2004; Milanesi et al., 2006). If is reduced in the sinoatrial node of rabbits with heart failure (Verkerk et al., 2003), whereas If is up-regulated in atrial and ventricular myocytes during heart failure in rats (Cerbai et al., 1996) and humans (Koumi et al., 1994, 1995; Cerbai et al., 1997; Hoppe and Beuckelmann, 1998; Hoppe et al., 1998). The abnormal automaticity that results from up-regulation of HCN channel expression may contribute to arrhythmia associated with heart failure (Opthof, 1998).
Reduction of If can have an antianginal effect, and ivabradine is currently used to treat ischemic heart disease (Vilaine, 2006). In addition, HCN channel blockers have been proposed as a novel treatment for atrial tachyarrhythmia. However, existing drugs are not very selective and have low potency. Understanding the molecular basis for drug block could enable the design of more potent and selective blockers.
ZD7288 is a substituted aminopyrimidinium compound (Fig. 1b, inset) with bradycardic and antianginal activity in experimental animals (BoSmith et al., 1993; Briggs et al., 1994). This compound potently reduces If in sinoatrial node cells but is a less potent blocker of Ih in neurons or heterologously expressed HCN channels. In sinoatrial node cells isolated from the guinea pig heart, ZD7288 slowed pacemaker activity at low concentrations (10 nM–1 µM) (Briggs et al., 1994) and blocked If with an IC50 of about 0.3 µM (BoSmith et al., 1993). In contrast, the IC50 for block of Ih was reported to be 2 µM in substantia nigra neurons of the guinea pig (Harris and Constanti, 1995), 41 µM for mouse HCN1 channels expressed in Xenopus oocytes (Shin et al., 2001), and 20 to 41 µM in transfected HEK293 cells expressing human HCN1, HCN2, HCN3, or HCN4 channels.
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). spHCN channel block is irreversible, whereas recovery from block of HCN1 is rapid. This difference in recovery kinetics was accounted for by three specific residues in the S6 domain of these channels (Shin et al., 2001). Together, these findings strongly suggest that the binding site for ZD7288 is located within the central cavity of HCN channels in a position interior to the activation gate, similar to many other blockers of voltage-gated ion channels. Using an Ala-scanning mutagenesis approach, we recently localized the drug binding site to residues in the S6 domain of hERG (Mitcheson et al., 2000), KCNQ1 (Seebohm et al., 2003), and Kv1.5 channels (Decher et al., 2004). Here, we use the same approach to characterize the binding site for ZD7288 on mouse HCN (mHCN) 2 channels expressed in Xenopus oocytes. Site-directed mutagenesis was used to change single residues located in the S6 domain and near the base of the pore helix of mHCN2. Reduced sensitivity of mutant channels to blockade by ZD7288 identified two residues important for interaction with the drug. Our findings suggest that specific residues located on the S6 domain of mHCN2 are among the molecular determinants of ZD7288 binding. |
Materials and Methods |
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). Human HCN (hHCN) 1 was a gift from D. Krafte (Icagen Inc., Durham, NC). Mutations were introduced into mHCN2 cDNA as described previously (Chen et al., 2000). Restriction mapping and DNA sequencing of the polymerase chain reaction-amplified segment were used to confirm the presence of the desired mutation and the lack of extra mutations. cRNA for injection into oocytes was prepared with SP6 Capscribe (Roche, Indianapolis, IN) after linearization with EcoRI. RNA quality was checked by gel electrophoresis, and concentration was quantified by UV spectroscopy and Ribogreen assay (Invitrogen, Carlsbad, CA).
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). In brief, stage IV and V X. laevis oocytes were injected with 10 to 50 ng of cRNA encoding WT or mutant mHCN channels. After injection with cRNA, the oocytes were cultured at 18°C for 1 to 3 days in Barth's solution containing 88 mM NaCl, 1 mM KCl, 1 mM MgSO4, 0.41 mM CaC12, 2.4 mM NaHCO3, 10 mM HEPES, 0.33 mM Ca(NO3)2, 50 µg/ml gentamicin, and 1 mM pyruvate, pH 7.4. For voltage-clamp experiments, oocytes were bathed in a modified ND96 external solution containing 96 mM NaCl, 4 mM KCl, 1 mM MgC12, 1 mM CaC12, and 5 mM HEPES, pH 7.6. Currents were recorded at room temperature (22–24°C) with a GeneClamp 500 amplifier (Molecular Devices, Union City, CA) using standard two-microelectrode voltage-clamp techniques (Stühmer, 1992). Current-voltage (I-V) relationships for WT and mutant channels were determined using 8-s test pulses applied from a holding potential of –30 mV to test potentials ranging from –130 to –30 mV. Pulses were applied once every 21 s. Deactivating (tail) currents were measured at +20 mV. To screen WT and mutant mHCN channels for their sensitivity to block by ZD7288, currents were elicited by repetitive 8-s pulses to –100 mV from a holding potential of –30 mV. Preliminary experiments using 40-s pulses indicated that 8 s was sufficient for WT mHCN2 currents at –100 mV to attain 94 ± 1% (n = 10) of its steady-state level. Drug-induced block of current was assessed by measuring inward current at the end of the 8-s hyperpolarizing pulse to –100 mV and calculating percent block based on currents measured before drug and after complete block of mHCN2 current with 20 mM CsCl.
ZD7288 (Tocris Bioscience, Ellisville, MO) was prepared as a 50 mM stock solution in distilled water. Cilobradine was provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT) and prepared as a 10 mM stock solution in distilled water. An aliquot of stock solution was dissolved in the modified ND96 external solution immediately before use to obtain the final desired drug concentrations.
Data Analysis. The time constants for HCN current activation (
act) at –100 mV and deactivation (deact) at +20 mV were determined using the standard exponential curve fitting routine of pClamp 8 software (Molecular Devices). Time-dependent activating currents were fitted with either one or two exponential functions:
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To quantify the voltage dependence of HCN current activation, the normalized tail current amplitude (Itail) measured at +20 mV was plotted versus test potential and fitted with a Boltzmann function (below) using Origin 7.5 software (OriginLab, Northampton, MA):
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To obtain the voltage required for half-maximal activation (V1/2) and the slope factor (k). The minimal open probability (min-Po) was defined as the minimal value of relative tail current.
Concentration-effect data were fit to the Hill equation, f = 1/{1 + (IC50/[D])nH}, where f is fractional block of HCN current, IC50 is the drug concentration required for 50% inhibition, [D] is the drug concentration, and nH is the Hill coefficient. Data are presented as mean ± S.E. (n = number of cells), and statistical comparisons between experimental groups were performed using the Student's t test. Differences were considered significant at p < 0.05.
Homology Model. A homology model of the pore of the S5-P-S6 region was developed based on crystal coordinates of KcsA (Doyle et al., 1998) using the PRIME module in Maestro (Prime, 1.0 edition; Schrödinger, Inc., Portland, OR). Sequence alignment was performed by first identifying potential transmembrane regions of the sequence spanning the S5-P-S6 region using Tmap (Persson and Argos, 1994) followed by anchoring conserved regions before alignment. The model derived was subjected to side chain refinement using PRIME to provide a starting template. S6 helix motion on activation was mimicked by mapping the intermediate states in 1° increments using Gly424 as a pivot from a closed reference state to an open state (Rajamani et al., 2005). The vector representing the motion from the closed to open state was determined by overlaying the crystal coordinates of KcsA and MthK, respectively (Jiang et al., 2002). Each intermediate state was subjected to a short simulated annealing protocol with harmonic restraints on the backbone heavy atoms (heating, 0.4 ps; equilibration, 0.6 ps; and dynamics, 5 ps; and harmonic constraint of 24 kcal/mol/Å2 was applied on the C
atoms) using the CHARMM force field (Brooks and Karplus, 1983; MacKerell et al., 1998). Giorgetti et al. (2005) recently reported a study of the pore region of HCN channels based on homology models. The study suggested that the bending amplitude of S6 helix upon gating was significantly smaller than that for the MthK S6 helix (Giorgetti et al., 2005); thus, an intermediate state was selected (12° twist compared with the closed reference state), and ZD7288 was docked into the pore region using standard protocols in Glide (Friesner et al., 2004). The poses generated based on the docking protocol were energy minimized and rank ordered by estimating their respective ligand-protein interaction energies. The computed electrostatic and van der Waals energy contributions in the bound and free states of the ligand were extracted to estimate the ligand-protein interaction energies. The electrostatic component was estimated using single point energies (RHF/3–21G*) computed in the presence and absence of protein environment represented as point charges (Frisch et al., 2004). The poses generated were then rank ordered based on their computed interaction energies, and the top-docked pose was used for data interpretation.
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Results |
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act fast = 433 ± 57 ms; act slow = 2.10 ± 0.23 s; n = 5). ZD7288 increased the time constants for both components of activation (act fast = 620 ± 70 ms; act slow = 3.29 ± 0.28 s; n = 5); however, the relative amplitude of the fast component of activation was not significantly altered (0.62 ± 0.04 in control and 0.67 ± 0.05 in the presence of drug). The time constant for current deactivation (deact) at +20 mV was 106 ± 2.8 ms in control and 89 ± 5.4 ms after 30 µM ZD7288 (n = 5). The amplitudes of tail currents measured at +20 mV were normalized to the peak value, plotted as a function of test voltage, and fitted with a Boltzmann function to obtain the isochronal voltage dependence of mHCN2 channel activation. ZD7288 reduced tail current amplitude and caused a negative shift in V1/2 (Fig. 1c). The average value for V1/2 was –73.4 ± 1.2 mV under control conditions and –82.7 ± 1.2 mV after addition of ZD7288 (n = 6). The slope factor of the activation curve was decreased from 7.2 ± 0.4 to 6.7 ± 1.0 mV in the presence of drug.
Drug-induced block of HCN channels can be potentially modulated by transmembrane voltage, the magnitude of inward current, or the state (open or closed) of the channel. To determine the effects of voltage on steady-state block, each oocyte was repetitively pulsed to a single test potential, and the block induced by 30 µM ZD7288 was assessed by measuring steady-state current at the end of the 8-s pulse. Drug-induced block of mHCN2 current was similar for test potentials between –130 and –110 mV but was greater at more positive potentials (Fig. 2a). The voltage dependence of block had an apparent valence (z
) of 5.6 as determined by fitting the fractional block of current versus test potential with a Boltzmann function.
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). Here, we show that block of WT mHCN2 by ZD7288 is also modulated by [K+]o (Fig. 2b). When oocytes were bathed in a solution containing 4 mM KCl, 30 µM ZD7288 reduced current by 71 ± 5% (n = 5). In contrast, when [K+]o was elevated to 96 mM, the same concentration of drug reduced the current by only 37 ± 3% (n = 4).
To explore the relative importance of state-dependent block, we examined the effect of ZD7288 on mutant channels with an impaired ability to close. We previously reported that specific mutations in the S4-S5 linker of mHCN2 disrupted the closed state (Chen et al., 2001). R318Q/Y331S mHCN2 channels are constitutively open (min-Po 1), and Y331A mHCN2 channels have a min-Po of 0.55 (Chen et al., 2001). The effects of ZD7288 and ZD7288 plus 5 mM CsCl on currents recorded from oocytes expressing WT or mutant mHCN2 channels are shown in Fig. 3. ZD7288 (30 µM) reduced the magnitude of WT mHCN2 channel current by 71 ± 5% (n = 5), R318Q/Y331S channels by 42 ± 2% (n = 4), and Y331A channels by 62 ± 5% (n = 4). Thus, an increase in min-Po was associated with decreased channel block, consistent with potency of block being related to the ability of a channel to close and trap a drug molecule within its inner cavity (Armstrong, 1971; Holmgren et al., 1997; Shin et al., 2001).
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Mutation of four residues (His397, Ile401, Ser420, Gly424) to Ala or Val yielded channels with immeasurable functional expression in oocytes, confirmed by lack of current reduction in response to addition of 20 mM CsCl to the external solution. A425V channels were also nonfunctional, but A425G channel currents were robust and similar to WT mHCN2. The biophysical properties of the 22 functional mutant channels were characterized by estimating the time constants for activation at –100 mV and deactivation at +20 mV. The voltage dependence of activation was determined from analysis of tail currents elicited at +20 mV following pulses to a variable test potential (Table 1). The shift in the V1/2 for channel activation ranged from +10 mV for I422A to –40 mV for A429V channels. Min-Po averaged 0.04 ± 0.01 for WT channel current (n = 10). For mutant channels, min-Po ranged from 0.02 for Y428A to 0.68 for I432A (Table 1).
The effects of 30 µM ZD7288 on the 22 mutant mHCN2 channels were determined. For this survey, currents were elicited by repetitive 8-s pulses to –100 mV from a holding potential of –30 mV. Only two mutations, A425G and I432A, significantly reduced block of current by 30 µM ZD7288 compared with WT channels (Fig. 4). Both residues are located on the S6 domain of HCN2, and a homology model based on KvAP indicate that they face toward the central cavity of the channel (Fig. 5).
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1 and yet can still be blocked by 42% with 30 µM drug.
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Cilobradine is another recently characterized HCN channel blocker (Van Bogaert and Pittoors, 2003; Stieber et al., 2006) that is about 3 times more potent than ZD7288. At a concentration of 10 µM, cilobradine inhibited WT mHCN2 channel current by 86 ± 2% (n = 5). In contrast, I432A and A425G channel currents were only reduced by 14 ± 1% (n = 4) and 19 ± 2% (n = 8), respectively, by this concentration of drug. The double mutant (I432A/A425G) channel was even less sensitive to 10 µM cilobradine (8 ± 2% inhibition; n = 4). Thus, both cilobradine and ZD7288 seem to interact with specific residues located on the S6 domain of mHCN2.
Hydrophobicity of Residue 432 in mHCN2 Determines Blockade by ZD7288. Ile432 of mHCN2 was mutated to three other hydrophobic residues, Phe, Val, and Leu. These mutations caused a variable negative shift in the voltage dependence of mHCN2 channel activation. The V1/2 values for activation curves were as follows: WT, –74 ± 1.3 mV; I432A, –85 ± 1.1 mV; I432F, –93 ± 0.4 mV; I432V, –95 ± 1.4 mV; and I432L, –78 ± 1.7 mV (n = 4–14). The slope factors for the activation curves varied from 7.7 ± 0.5 mV for WT and 10.3 ± 0.5 mV for I432A mHCN2 (Table 1). ZD7288 (30 µM) blocked WT, I432L, I432F, and I432V mutant channels at a test potential of –100 mV to a similar extent (Fig. 7a), and these mutant channels were more sensitive to drug than I432A channels (Fig. 6b). Substitution of Ile432 with Leu, Val, or Phe increased the IC50 by 1.46, 2.29, and 2.72-fold relative to WT mHCN2 channels (Fig. 7b). Based on an extrapolation of the concentration-effect relationship, the IC50 for I432A channels was estimated to be 150-fold greater than WT channels. The rank order of IC50 values was the inverse of the rank order for a measure of hydrophobicity (cyclohexane/water distribution coefficient) of the substituted amino acid: Ala >> Phe > Val > Leu = Ile. Thus, highly hydrophobic residues such as Ile, Leu, and Val favor block, whereas mutation to Ala reduced block.
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Differential Sensitivity of HCN1 and HCN2 Channels to ZD7288. The sequence of hHCN1 is identical to mHCN2 in the region subjected to Ala-scanning mutagenesis, with one exception; the residue equivalent to Ile432 in mHCN2 is a Val (Val390) in hHCN1. hHCN1 channel current was about 5 times less sensitive than mHCN2 channel current to block by ZD7288 (Fig. 8a). Therefore, we mutated Val390 of hHCN1 to Ile and Ala and assessed the effects of ZD7288 on the resulting mutant channels. These mutations in hHCN1 had only minor effects on channel activation. The V1/2 for activations were –69 ± 1.1 mV (k = 8.9 ± 0.6 mV) for WT hHCN1, –63 ± 0.4 mV (k = 10.7 ± 0.6 mV) for V390I, and –72 ± 2.1 mV (k = 13.8 ± 0.8 mV) for V390A channels (n = 8 for each channel type). The rate of channel activation at –120 mV was not altered by the V390I mutation (act = 67 ± 4 ms) compared with WT hHCN1 (act = 69 ± 4 ms). V390A channels activated more slowly at –120 mV (act = 287 ± 18 ms). As predicted from results obtained by mutagenesis of mHCN2, V390I hHCN1 channels were more sensitive and V390A hHCN1 channels were less sensitive to block by ZD7288 (Fig. 8, b and c). Thus, the difference in sensitivity to ZD7288 between HCN1 and HCN2 channels can be largely accounted for by a single residue (Val versus Ile) in the S6 domain.
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Discussion |
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; Stieber et al., 2006). Block of pacemaker current in the heart is used to treat angina (Vilaine, 2006) and could theoretically have antiarrhythmic activity in heart failure, a condition where HCN channel expression is up-regulated and could induce abnormal ventricular automaticity (Cerbai et al., 1996). However, existing HCN blockers are characterized by low potency and lack of specificity and can be proarrhythmic (Stieber et al., 2006). A better understanding of the molecular mechanisms of HCN channel block and the structural basis of the drug binding site could facilitate the discovery of more potent and specific agents.
Shin et al. (2001) previously characterized the mechanism of mHCN1 and spHCN block by ZD7288. They found that the drug required channel opening before block could occur and that the drug could be trapped inside the pore when the channel was subsequently closed by membrane depolarization. Moreover, the voltage dependence of mHCN1 block by 100 µM ZD7288 was steep, with a z of 4.2. Using a different pulse protocol, we also found a steep relationship for block of mHCN2 channels by ZD7288 (z = 5.6). As discussed by Shin et al. (2001), such a steep voltage dependence for drug block cannot be caused by the intrinsic voltage dependence of the positively charged blocker and is more likely due to preferential binding of ZD7288 to the closed state of HCN channels (accessed after channel has opened) or to block of two open states (O1 and O2) with different affinities. A recent study of native Ih in rat hippocampal CA1 pyramidal neurons identified two open states for native HCN channels with mean open times of 3.2 and 30 ms at –100 mV (Simeone et al., 2005). ZD7288 reduced the mean open times for both open states at potentials negative to –80 mV and prolonged the latency to first channel opening. However, it is still unclear if ZD7288 preferentially blocks the closed or open state of HCN channels. The finding that block of HCN2 channels by ZD7288 was decreased when [K+]o was elevated from 4 to 96 mM suggests that voltage-dependent block may be caused, at least in part, by an increased influx of permeant ion that relieves channel block, similar to the [K+]o-dependent clearing of tetraethylammonium from the inner pore of delayed rectifier K+ channels in squid axon (Armstrong, 1971). Alternatively, the change in channel conductance associated with elevated [K+]o may cause a structural rearrangement that decreases the affinity of drug binding.
Mutagenesis identified Ala425 and Ile432 as residues of the mHCN2 channel pore that interact with ZD7288. An HCN2 homology model based on KcsA (Fig. 5a) predicts that the side chains of both residues face toward the central cavity of the HCN2 channel. The top-docked pose selected for ZD7288 highlights a key interaction component with the charged ring aligning with the axis of the central cavity (Fig. 5b), closely corresponding to the localization of K+ ions as seen in the KcsA crystal structure (Doyle et al., 1998). The phenyl ring of the drug occupies a hydrophobic cavity formed by the residues of interest Ala425 and Ile432 (Fig. 5c). Based on the model, Ile432 forms the hydrophobic base of the pore with Ala425 contributing to the observed hydrophobic contact surface. Mutation of Ala425 to Gly increased the IC50 by 10-fold. This finding is consistent with the docked pose, which suggests that the drug may interact with the methyl side chain of this residue. However, it is also possible that mutation to Gly altered the positioning of nearby residues that decreased binding affinity. The evidence for the importance of Ile432 as a binding residue is more convincing because multiple mutations decreased channel sensitivity. From the docked pose, modeling suggests a greater impact arising from mutation of Ile432 to Ala due to loss of substantial contact surface area. We also found that Ala425 and Ile432 are important for normal block of mHCN2 channels by cilobradine. However, because we did not test all the S6 mutant channels with cilobradine, it is uncertain whether both drugs interact with a common or merely overlapping binding site. A previous study had suggested, but did not prove, that ZD7288 might bind to a residue homologous to Ile432 in the S6 domain of mHCN1 (Val359) and spHCN (Ile460) channels (Shin et al., 2001). Block of spHCN by ZD7288 is irreversible, but block of mHCN1 channels is readily reversible. Reversibility of block of spHCN could be induced by mutation of three residues in S6 (F456Y, L458M, and I460V) to match the S6 sequence of mHCN1 channels (Shin et al., 2001). In hERG K+ channels, Phe656 is located in a homologous position as Ile432 in mHCN2 and is also an important residue for drug binding (Mitcheson et al., 2000). Similar to our findings for mHCN2, mutations that decrease the hydrophobicity of amino acid 656 in hERG cause a decrease in sensitivity to blockers (Fernandez et al., 2004).
Limited contacts of ZD7288 with the pore region of the HCN2 channel could explain the low potency of the compound. However, as is the case for all Ala-scanning mutagenesis studies, it is also conceivable that more or different interaction residues would have been detected if other amino acid substitutions were made and characterized. Another limitation of the approach is that not all Ala mutations produced functional channels; therefore, important binding residues (e.g., at the base of the pore helix) may have gone undetected.
In summary, using an Ala-scanning approach, we have mapped the binding site for ZD7288 on HCN2 channel subunits to the midregion of the S6 domain. We suggest that hydrophobic interactions between ZD7288 and one or more Ile432 or Ala425 residues are responsible for block of the homotetrameric HCN2 channel.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HCN, hyperpolarization-activated, cyclic nucleotide-modulated; ZD7288, 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride; spHCN, sea urchin sperm HCN; mHCN, mouse HCN; hHCN, human HCN; WT, wild type; I-V, current-voltage; cilobradine, [(S)-(+)-1,3,4,5-tetrahydro-7,8-dimethoxy-3-((1-(2-(3,4-dimethoxyphenyl)-ethyl)-3-piperidinyl)-methyl)-2H-3-benzazepin-2-one-hydrochloride]; hERG, human ether-a-go-go-related gene.
Address correspondence to: Michael C. Sanguinetti, Department of Physiology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, 95 South 2000 East, Salt Lake City, UT 84112. E-mail: sanguinetti{at}cvrti.utah.edu
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