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Originally published In Press as doi:10.1074/jbc.M705835200 on September 6, 2007

J. Biol. Chem., Vol. 282, Issue 44, 31972-31981, November 2, 2007
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Activation Gating of hERG Potassium Channels

S6 GLYCINES ARE NOT REQUIRED AS GATING HINGES*Formula

Rachael M. Hardman{ddagger}, Phillip J. Stansfeld§, Sarah Dalibalta{ddagger}, Michael J. Sutcliffe, and John S. Mitcheson{ddagger}1

From the {ddagger}Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom, the §Structural Bioinformatics and Computational Biochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, and the Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7ND, United Kingdom

Received for publication, July 16, 2007 , and in revised form, August 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The opening of ion channels is proposed to arise from bending of the pore inner helices that enables them to pivot away from the central axis creating a cytosolic opening for ion diffusion. The flexibility of the inner helices is suggested to occur either at a conserved glycine located adjacent to the selectivity filter (glycine gating hinge) and/or at a second site occupied by glycine or proline containing motifs. Sequence alignment with other K+ channels shows that hERG possesses glycine residues (Gly648 and Gly657) at each of these putative hinge sites. In apparent contrast to the hinge hypotheses, substitution of both glycine residues for alanine causes little effect on either the voltage-dependence or kinetics of channel activation, and open state block by intracellular blockers. Substitution of the glycines with larger hydrophobic residues causes a greater propensity for the channel to open. We propose that in contrast to Shaker the pore of hERG is intrinsically more stable in the open than the closed conformation and that substitution at Gly648 or Gly657 further shifts the gating equilibrium to favor the open state. Molecular dynamics simulations indicate the S6 helices of hERG are inherently flexible, even in the absence of the glycine residues. Thus hERG activation gating exhibits important differences to other Kv channels. Our findings indicate that the hERG inner helix glycine residues are required for the tight packing of the channel helices and that the flexibility afforded by glycine or proline residues is not universally required for activation gating.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Potassium (K+) channels are integral membrane proteins that form a pore for conduction of K+ ions through the lipid bilayer membrane. They have evolved to perform a wide range of physiological processes and share a similar overall structure consisting of four subunits, which co-assemble to form a central pore coupled to additional regulatory domains that detect a huge variety of different signals. hERG (human ether-à-go-go-related gene) belongs to the voltage gated family of potassium (Kv)2 channels. It is widely expressed in the heart and nervous system. It may also be ectopically expressed in certain types of cancer (reviewed in Ref. 1). The physiological importance of hERG is illustrated by the discovery that block of the pore by medications or loss of channel function due to inherited mutations carries an increased risk of sudden cardiac death (2). Therefore, there is considerable interest in gaining further insight into the structural basis of hERG gating and drug binding (3, 4).

In recent years, crystal structures have provided tremendous insight into how K+ channels function. The KcsA and Kir-Bac1.1 structures correspond to channels in the closed conformation (5, 6). The inner helices of the pore that extend across the membrane and line the inner cavity are straight and come together to form a right-handed helical bundle constricting the channel at the intracellular entrance to the inner cavity, thus presenting a barrier to K+ movement. In contrast, MthK (7, 8), KvAP (9), and Kv1.2 (10) have all been crystallized in the open state. All these channels display a bend in the inner helices, which splays the C-terminal (intracellular) end of the inner helix away from the central axis of the pore to create a large aperture at the intracellular mouth of the channel.

The inner helices appear to bend at a position, which is highly conserved as a glycine in K+ channels (Fig. 1). This led to the proposal that the glycine residue forms a kink or gating hinge that is required for the opening of the activation gate (7). An important feature of glycine is its ability to introduce conformational flexibility into protein structures [e.g. (11)]. This can occur through destabilization of {alpha}-helices (although this propensity is not as strong in non-aqueous environments such as transmembrane helices) and because the small side chain of glycine permits freedom of rotation about the phi and psi dihedral angles of amino acids that enables backbone conformations to be adopted that are not sterically tolerated by other amino acids (e.g. Ref. 12). The apparent importance of this glycine residue is supported by several mutational studies. Replacing it with alanine produces non-functional channels or shifts the closed-open equilibrium toward the closed state (13, 14), whereas substitution to proline increases open probability (13–15), perhaps due to insertion of a kink that favors the open conformation (16).

S6 mutations in Shaker that perturb activation gating cluster either in the vicinity of the glycine gating hinge or near where the helices bundle together (17), in close proximity to the Pro-X-Pro motif found in many Kv channels. The amide of proline cannot act as a hydrogen donor and thus prolines disrupt the backbone hydrogen bond network that stabilize {alpha}-helices. Mutagenesis and in-silico simulation studies have indicated that the {alpha}-helix destabilization conferred by prolines is required for gating (11, 18, 19). The Pro-X-Pro motif is not fully conserved (Fig. 1A), but many K+ channels also have another glycine in the analogous position to the second proline that may perform a similar function (20).

The experimental results from a number of K+ channel studies suggest a common theme in which glycines and prolines on the inner helices are required for bending during channel gating. However, the assertions that these function as hinge points and are universal to ion channels are not supported by the observation that in some channels substituting for alanine, a residue that stabilizes {alpha}-helices (21, 22), has little effect on channel gating (23, 24). hERG K+ channels have two glycines on S6. Gly648 aligns with the putative glycine gating hinge and Gly657 is in the analogous position to the second proline in the Pro-X-Pro motif in Kv channels. In a previous substituted alanine scan of S6 we found that mutation of either of these glycine residues had little effect on the voltage and time-dependent kinetics of activation and deactivation (24). In the present study we find that substitutions to small residues in hERG, in contrast to other channels, have minimal effects on gating, whereas substitutions to bulky, hydrophobic residues cause stabilization of the open state. Molecular dynamics simulations suggest that the S6 {alpha}-helices of hERG display an inherent flexibility that is sufficient for activation gating. Our results indicate that S6 glycines are not required as gating hinges but the small side chain enables tight packing within the pore of the channel that is essential for normal activation gating.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Biology and Oocyte Injection—Site-directed mutagenesis was performed using the QuikChange mutagenesis technique (Stratagene, La Jolla, CA). The wild-type (WT) background used was hERG with a silent mutation introducing a SalI restriction site within the S2 encoding sequence (a gift from Dr Harry Witchel, University of Bristol). A DNA fragment from SalI to XhoI, encoding S2-S6 of hERG, was subcloned into pBluescript KS II, mutagenesis carried out by PCR and the fragment religated into pSP64 hERG for expression in Xenopus oocytes. All mutations were verified by sequencing. Vector DNA was linearized with EcoRI and in vitro transcription was performed using SP6 RNA polymerase (mMessage mMachine, Ambion, Austin, TX). Xenopus laevis oocytes were isolated, defolliculated, maintained in culture and injected with wild type or mutant cRNA as previously described (25).

Electrophysiology—Whole cell currents were recorded in Xenopus oocytes using two electrode voltage clamp, as previously described (26). Microelectrodes were filled with 3 M KCl and the tips broken to give resistances of 1.1–1.5 M{Omega}. Recordings were made at room temperature 1–5 days after cRNA injection. Data were low pass filtered at 1 or 2 kHz sampled at 5 kHz and saved to computer for off-line analysis using a digidata 1320A data acquisition system (Molecular Devices, Sunnyvale, CA). Oocytes were perfused with low chloride, MES-based solutions to attenuate endogenous chloride currents (27). The 2 mM K+ extracellular solution contained (in mM) NaMES 96, KMES 2, Ca(MES)2 2, MgCl2 1, HEPES 5, pH 7.6. To record from mutants with a negatively shifted voltage dependence of inactivation a high-K+ solution was used (24, 28), consisting of (in mM) NaMES 2, KMES 96, Ca(MES)2 2, MgCl2 1, HEPES 5, pH 7.6. The properties of mutants were directly compared with WT hERG recorded in the same extracellular solution. All bath solutions were applied using a switching device to enable rapid exchange of solutions (27).

Voltage Protocols and Data Analysis—The holding potential was –90 mV for WT hERG, but was adjusted to more negative potentials depending on the voltage-dependent properties of different mutants. Details of protocols are provided in the figure legends. Analyses of the kinetics for activation and deactivation were performed using Clampfit software (Molecular Devices). The decay phase of tail currents were fit with the double exponential function in Equation 1,

Formula 1(Eq. 1)
where {tau}fast and {tau}slow are the time constants of the fast and slow components of deactivation, Afast and Aslow are the amplitudes of each component and C is a constant. Time constants for activation at 0 mV were investigated with an envelope of tails protocol. Membrane potential was stepped to 0 mV for varying time periods of between 50 and 1500 ms, and hERG current activation monitored with measurements of peak tail current amplitude upon repolarization to –70 mV. Peak tail current amplitudes were plotted against pulse duration and fitted with a single exponential function. Parameters for the voltage dependence of activation were obtained by fitting peak tail current-voltage relationships with the Boltzmann function in Equation 2,

Formula 2(Eq. 2)
where G(V) is normalized conductance, Vm is the test potential, V0.5,act is the mid-point, and k the slope factor for activation. Free energies for work in opening channels were calculated as {Delta}G =–ZFV0.5,act, where Z is an estimate of effective gating valence and is derived from k = RT/ZF, with the constants RT/F having a value of 25 mV at room temperature. Perturbation energies for the effects of mutations on activation gating were calculated according to Equation 3 (14, 17).

Formula 3(Eq. 3)
Data are presented as mean ± S.E. (n = number of cells). Statistical comparisons were performed using the unpaired Student's t test where appropriate. Differences were considered significant at p ≤ 0.05.

Channel Block—Open channel block was investigated with terfenadine, a high affinity blocker of hERG that binds within the inner cavity and retains its binding affinity for G648A hERG (24). Terfenadine (Sigma) was prepared daily by dilution to the required concentration from a 10 mM stock in dimethylsulfonic acid. Protocols for investigating state-dependent block of G648A/G657A hERG are described under "Results." For all other mutants, inhibition was measured with long (5 s) depolarizing pulses to 0 mV applied every 6 s, with only short (400 ms) repolarizations to elicit tail currents, thus ensuring that channels are primarily in the open/inactivated state. Peak tail currents following steady-state inhibition by terfenadine were normalized to control and the corresponding concentration-response relationships fitted with a Hill function to obtain IC50 values (see (28)).


Figure 1
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  		FIGURE 1.
Mutating S6 glycine residues to alanine gives functional channel expression. A, sequence alignment for the Pore helix (P) through to the S6 transmembrane helix of hERG (SWISS-PROT ID: Q12809), and hEAG (O95259), dEAG (Q02280), Shaker (P08510), Kv1.2 (P63142), KvAP (Q9YDF8), MthK (O27564), KcsA (P0A334), and Kir2.1 (P63252). The two hERG glycine residues are marked by arrows above the sequence alignment, and black bands on the gray cylinders that represent the proposed {alpha}-helices. The location of the Pro-X-Pro motif is indicated by a black box. The relative positions of the pore helix, selectivity filter (SF), and S6 are illustrated in the scheme below the sequence alignments. B, schematic representation of the pore domain. Only two diagonally opposed subunits are shown. The highly conserved SF is indicated by short lines representing each carbonyl oxygen that participates in K+ ion coordination. C–F, representative currents from wild type (C and E), G657A (D), and G648A (F) hERG channels. 5-s test pulses were applied to potentials between –70 and +50 mV from a holding potential of –90 mV. Currents in C and D were recorded in 2 mM K+ extracellular solution, with a tail current potential of –70 mV. Currents in E and F were recorded in 96 mM K+, with a tail current potential of –90 mV. G and H, mean normalized data for voltage dependence (G) and time course (H) of activation. I and J, voltage dependence of slow (I) and fast (J) time-constants ({tau}) of deactivation (note {tau} values are plotted on a log10 scale). Alanine substitution for either glycine gave functional channels whose voltage dependence and activation kinetics were relatively similar to WT hERG channels.

 
Molecular Modeling and Dynamics Simulation—Homology models of hERG (SWISS-PROT (29) ID: Q12809 [GenBank] ) were created from the KcsA crystal structure (PDB (30) ID: 1K4C [PDB] (31)) using Modeler 7v7 (32). The KcsA structure was chosen rather than KvAP or Kv1.2 because we wanted to initiate the simulations from a closed state model. All four subunits were modeled in 4-fold rotational symmetry incorporating the region of hERG from Tyr611, at the N-terminal end of the Pore helix, to Arg666, at the C-terminal end of the S6 transmembrane helix (Fig. 1B). Mutations were performed in Pymol (33). S5 and the turret were not included in the simulations because they made the models unstable; in addition, the structure of the turret (which is much longer than the template structures) and precisely how it packs against the rest of the pore is currently uncertain.

The homology models of hERG were placed into a POPC membrane (34). Voidoo and Flood were used to probe and add waters to the central cavity of the channel (35). K+ ions were placed in the channel at sites S0, S2, S4, and Scav, using the crystallographic coordinates from KcsA, with water molecules positioned at S1 and S3 of the selectivity filter (31, 36). Water molecules were also positioned behind the backbone of the selectivity filter, as observed in KcsA (1K4C). The system was fully solvated above and below the membrane, interspersed with chloride ions to neutralize the system. The periodic box surrounding the ~17,000 atom system was ~6 x 6 x 6.5 nm3.

Molecular dynamics simulations were performed with Gromacs (v3.2) (37) using a modified version of the GROMOS96 43a1 force field. Simulations used semi-isotropic pressure coupling, while the temperatures of the lipid, protein and solvent were coupled separately to an external bath held at 300 K (38). The simple point charge (SPC) model was used for water (39). The LINCS algorithm was used to constrain bond lengths (40). Partial Mesh Eswald (PME) was used to handle electrostatic interactions, while Cut-off parameters were used for Van der Waal's interactions (VdW).

The system was energy-minimized and then simulated for 200 ps of restrained molecular dynamics, during which the non-hydrogen protein atoms and potassium ions were restrained. After the initialization steps, the system was subjected to 20 ns of unrestrained molecular dynamics, during which coordinates were saved every 10 ps for analysis.

Swivel and Kink Analysis—The program SWINK (16) has been used previously to measure the kink and swivel of {alpha}-helices in other channels and domains (41). In this study, the S6 helices of hERG were extracted from the simulations, with the snapshots of the helices aligned at the N-terminal end. The kink and swivel angles of the helices were then calculated for the simulated S6 helices of hERG, and compared with angles observed for the inner helices of the KcsA (M2) and KvAP (S6) crystal structures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Alanine Substitution of S6 Glycine Residues Has Little Effect on hERG Channel Function—It has been suggested previously that there are two potential sites where the inner helices (S6) could bend to allow channel opening (6, 7). Sequence alignment (Fig. 1A) shows a highly conserved glycine residue that is postulated to be the gating hinge for many K+ channels. In Kv channels a partially conserved Pro-X-Pro motif located 7 residues downstream of this first glycine has also been proposed as a site for the flexibility that is required for the movement associated with channel gating. In hERG, and many other ion channels, the residue in the position of the second proline of this motif is a glycine. To investigate if either of these glycines in hERG (Gly648 and Gly657) are required for normal channel gating they were individually substituted for alanine (a residue that stabilizes {alpha}-helices (21, 22)), and the activation and deactivation properties compared with WT hERG.

Alanine substitution of Gly657 had only minor effects on the activation gating of hERG (Fig. 1 and Table 1). The V0.5,act was shifted by only –5.7 mV relative to WT hERG, and the activation time constants at 0 mV changed from 228 ± 12 ms for WT hERG to 175 ± 7 ms for G657A. On the other hand, deactivation at potentials between –70 and –90 mV was very significantly faster (Fig. 1, I and J). Thus the kinetics of channel opening were relatively unaffected by substituting Gly657, whereas channel closure was more rapid.


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  		TABLE 1
Activation and deactivation properties of WT and mutant hERG channels

V0.5,act values and slope factors were obtained from fitting tail current-voltage relationships with a Boltzmann function (see "Experimental Procedures"). Activation time constants were derived from a single exponential fit to plots of tail current amplitude versus test pulse duration. Deactivation time constants and the relative proportion of slow deactivation (As/Af + As) were determined from double exponential fits to the decay of tail currents measured at –90 mV. n, the number of cells analyzed. N.E. indicates mutants that failed to display ionic currents while N.D. relates to protocols that were not performed on expressing mutants. Extracellular recording solutions contained either 2 or 96 mM K+ as indicated. Statistical comparisons were made against WT hERG measurements under identical recording conditions.

 
G648A hERG currents (Fig. 1F) had to be recorded in high-K+ solutions because of a negative shift in the voltage dependence of C-type inactivation (24). Nevertheless, compared with WT hERG in the same recording conditions the effects on the voltage and time dependent kinetics of activation and deactivation were relatively minor. The relative lack of effects on hERG activation gating are not consistent with a critical role for Gly648 in gating and are in contrast to the effects of alanine substitutions at analogous positions in Shaker and Kv7.2 channels, in which a total loss of function is observed (14, 17, 42).

To test if flexibility associated with the remaining single glycine in the G648A and G657A mutants was sufficient for relatively normal activation gating, we investigated the gating properties of the double mutant G648A/G657A. If the flexibility of a single glycine is required for normal activation gating then removing both glycines would be expected to substantially reduce open probability by slowing activation or shifting it to more positive potentials. However, a negative shift of V0.5,act of 7.2 mV, with little change in the time course of activation was observed (see supplemental Fig. S1). These results suggest that hERG channels may not require the flexibility conferred by glycine residues to gate effectively with respect to K+ permeation.


Figure 2
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  		FIGURE 2.
Mutation of Gly648 hERG to larger residues destabilizes the closed state. A, representative currents elicited by I-V (left) and fully-activated I-V (right) protocols when Gly648 is mutated to Val, Ile, or Leu. All recordings were made in 96 mM K+ using a holding potential of –120 mV. For I-V protocols, tail currents were measured at –140 mV following 5-s pulses between –120 and +40 mV. B–E, mean normalized data showing voltage dependence and kinetics of channel gating. G648V and G648I both displayed strong hyperpolarizing shifts in the voltage dependence of activation (B) while all 3 mutants showed faster activation kinetics (C) and drastically slowed deactivation (D and E).

 
Although substitution of both glycines did not alter gating with respect to K+ it remained possible that S6 flexibility was sufficient to create an aperture large enough for K+, but nevertheless greatly reduced compared with WT hERG. Another scenario we considered was that loss of S6 flexibility resulted in the intracellular aperture being permanently open and that the selectivity filter mediated gating with respect to K+; the selectivity filter acting as a gate has been proposed for inward rectifier, cyclic nucleotide gated channels (43, 44) and recently for the low open probability of KcsA channels (45). A prediction from the first model is that large channel blockers would be unable to enter the inner cavity and open state block would be abolished. In contrast, the second model would predict that pharmacological block would occur in either the open or closed states because the S6 helices would no longer act as a barrier to binding of the blocker within the inner cavity. Therefore state-dependent block of G648A/G657A was tested with terfenadine, a potent blocker of hERG with a minimum diameter of 10 Å. Gly648 and Gly657 mutants retain their sensitivity to block by terfenadine (see Table 2). Application of 10 µM terfenadine to channels in the open state produced a time-dependent reduction in current amplitudes that was partially reversed upon wash-off (supplemental Fig. S2). In contrast, application of terfenadine while channels were kept predominantly in the closed state had no effect on current amplitudes. These results indicate that, as for WT hERG, the intracellular gate formed by S6 still functions to gate access of terfenadine to the inner cavity of G648A/G657A hERG channels in the open state only. Because terfenadine is 10 to 20 Å in diameter, the activation gate of G648A/G657A hERG must open at least 10 Å to permit access of the drug to the inner cavity, indicating that S6 glycines are not absolutely required as gating hinges during activation gating.


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  		TABLE 2
Block of WT and mutant hERG channels by terfenadine

Mean percent inhibition of WT and mutant channel currents by either 3 or 10 µM terfenadine. Most mutations did not significantly alter channel block.

 
Proline Mutants Are Unable to Fully Close—Proline residues in {alpha}-helices tend to introduce a kink with an average angle that is similar to the 25° bend observed at the glycine hinge for the crystal structures of channels in the open state (7, 9, 11, 21). Studies on several different ion channels, in which the putative glycine hinge has been replaced by proline, have resulted in channels that favor the open state (15, 17). Mutation of Gly648 or Gly657 also significantly altered the gating properties of hERG. G648P gave large terfenadine-sensitive inward currents at the –90 mV holding potential, which increased in amplitude at more hyperpolarized potentials. This indicates that a proportion of channels were unable to fully deactivate. With depolarization there was further channel activation with a V0.5,act of –66.1 ± 0.8 mV. Fast and slow time constants for deactivation were also significantly slower than WT hERG (Table 1). For G657P, the effects on channel gating were even more pronounced. Current amplitudes were small at depolarized potentials because of channel inactivation. Stepping to negative potentials resulted in rapid recovery from inactivation to give large sustained inward currents that increased linearly with K+ driving force and that showed no deactivation for prolonged periods of time at potentials as negative as –180 mV. Examples of G648P and G657P currents are given in supplemental Fig. S3. Percentage inhibition of G657P hERG by 10 µM terfenadine was similar to WT hERG (Table 2). Thus, although G657P channels were unable to close, the binding site for terfenadine was not substantially altered. This suggests that the inner helices of G657P channels are locked in a conformation that is similar to the open conformation of WT hERG, at least with regard to residues close to Gly657 (Tyr652 and Phe656) that form the drug binding site. In summary, prolines at positions 648 and 657 prevent hERG channels from closing normally and favor channel opening.


Figure 3
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  		FIGURE 3.
Mutation of Gly657 hERG to larger residues stabilizes the open state. A, representative currents from the mutants G657V and G657I using I-V (left) and fully activated I-V (right) protocols. All recordings were made using the same conditions as those in Fig. 2, B and C, mean normalized data showing voltage-dependence and kinetics of channel gating. Both mutations showed strong hyperpolarizing shifts in the voltage dependence of activation (B) with faster activation kinetics (C). Time constants of deactivation were similar to WT hERG (Table 1).

 
Are S6 Glycines Required for Packing?—In addition to conferring flexibility, the small size of glycine residues is known to be important for close packing of {alpha}-helices (46). In homology models of hERG based on channel structures crystallized in the open or closed state it is notable that Gly648 is closely packed against the selectivity filter. Interestingly, G648A does not significantly alter activation gating but does alter inactivation, consistent with effects of the larger alanine on the open stability of the selectivity filter. Mutation of Thr623 to alanine on the C-terminal end of the pore helices of hERG has an identical effect on hERG channel gating, suggesting that residues at 648 and 623 are positioned close together (24). To investigate the possibility that the small side chain volume of glycine is required for normal packing of the inner helices we substituted the glycines for amino acids with larger side chain volumes.

Functional currents were observed when Gly648 was replaced by Val, Leu, and Ile (Fig. 2A). Large negative shifts in the voltage dependence of activation of 29 and 35 mV were observed for G648V and G648I respectively (Fig. 2B), and the fast and slow time constants for deactivation were significantly slowed (Fig. 2, D and E). Although, G648L activation parameters were not significantly different from WT hERG, as with all other mutants deactivation was considerably slower (see G648S and G648P data in Table 1). There was also little change to channel block by terfenadine (Table 2). These results suggest that rather than reducing S6 flexibility, which would reduce open channel block, slow activation and shift it to positive potentials, these glycine substitutions do the reverse and prevent the channel from closing normally.

Negative shifts in the V0.5,act for the 657 mutants were even more pronounced than those for the 648 mutants (Fig. 3). G657V and G657I produced negative shifts of 52 and 54 mV respectively. Unlike the Gly648 mutants, there were no significant effects on deactivation time constants (Table 1), but activation time constants were significantly faster compared with WT hERG (Fig. 3C), decreasing from 240 ± 18 ms for WT hERG to 126 ± 22 ms and 129 ± 3 ms for G657V and G657I, respectively.

Analysis of amino acid substitution frequencies in proteins reveals that glycines are most commonly substituted for alanine or for serine (47). Substitution of Gly648 and Gly657 with serine resulted in significant changes in gating properties. G648S deactivation was dramatically slowed and G657S activation was shifted to negative potentials by 30 mV. These results are characteristic of the Val, Ile, and Leu mutants, and indicate that replacing Gly with another polar residue does not result in normal gating. These findings provide further evidence that it is amino acid size, rather than polarity, that contributes to the effects on channel gating. Thus, our data are more consistent with glycines being required at positions 648 and 657 for close packing rather than S6 flexibility.

To identify the residues that are packed against positions 648 and 657, we made a series of double mutants to investigate if normal channel function of G648V and G657V could be rescued by decreasing the size of residues that, based on our homology models of hERG, were predicted to pack against these side chains. The models suggest that Leu622 and Thr623 (located at the C-terminal end of the pore helix) pack against Gly648, while Phe656, Asp658, and Ser660 from neighboring subunits are predicted to be in close proximity to Gly657. However, the double mutants either failed to express, suggesting a disruption in folding, or retained the negative shift of activation exhibited by the single glycine mutants (Table 1). The inability to rescue channel function could arise from choosing the wrong amino acid partner or because the 648 and 657 side chains are packing against the peptide backbone, which would not be influenced by these mutations.

Do S6 Mutations Alter the Energetics for Channel Activation—Previous work by Yifrach and MacKinnon (17) has shown that mutations in the pore of Shaker cause alterations in the free energy of activation gating in two separate locations; the inner helix bundle crossing (corresponding to the region around Gly657 in hERG) and where S6 is packed against the pore helix (close to Gly648). We calculated the changes in free energy ({Delta}G) for a number of hERG channel S6 mutations using the equation ZFV0.5,act where V0.5,act is the mid-point of activation, Z is the effective charge derived from the slope of the Boltzmann fits to the activation data and F is Faraday's constant. Perturbation energies ({Delta}{Delta}G) provide a means for comparing the energetics of activation gating of different S6 mutations and were calculated using {Delta}{Delta}G ={Delta}Gwt –{Delta}Gmut. Most mutations caused a relatively small perturbation in gating (data not shown). However, it was notable that the largest perturbation energies were seen with mutations at Gly648 and at positions 657, 658 and 659. In contrast to Shaker and mammalian Kv1.5 channels, in which the work required to open channels is increased by mutations at putative hinge positions (14, 19), mutation of Gly648 and Gly657 to large residues caused pronounced negative shifts in {Delta}{Delta}G (e.g. G657V, supplemental Table S1), indicating that these mutations facilitate channel opening. Nevertheless, perturbation energies are only a crude measure of pore equilibrium energies since other gating processes (e.g. movement of the voltage sensor and coupling between the voltage sensor and pore domains) are also included in {Delta}{Delta}G. For several mutants, the slopes of the activation curve as well as the V0.5 were effected, indicating complex effects on voltage dependent gating. This is likely to explain the relatively poor correlation between {Delta}{Delta}G and residue size. An analysis focusing on gating parameters was more revealing (Fig. 4). For Gly648 mutants there was a good correlation between residue size and deactivation time constants, suggesting that the increase of side chain volume stabilizes the open state. In the case of Gly657 mutants, there were good correlations with activation parameters (V0.5,act and {tau} values) suggesting that large residues in this position destabilize the closed state.


Figure 4
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  		FIGURE 4.
Correlations between side chain volume and gating properties. A and B, relationships between side chain volume and deactivation for residues at position 648 (A) and V0.5,act at position 657 (B) are shown. Values for amino acid side chain volumes are taken from Zamyatnin (56). Results for deactivation at –90 mV or V0.5act were plotted against side chain volumes and analyzed using Pearson's correlation. For position 648, r2 values of 0.93 and 0.74 were obtained for correlations with the slow and fast components of deactivation respectively. G648S data points were considered as outliers and were excluded from the regression fits. For position 657, the r2 value was 0.90. The identity of the relevant residue is indicated by a single letter code next to each data point.

 
Molecular Dynamics Simulations of S6 Flexibility—Computational approaches such as molecular modeling and simulation are valuable tools for extrapolating from available crystal structures to unsolved proteins and for investigating conformational dynamics necessary for protein function based on static crystal structures. To investigate the flexibility associated with gating in the pore of hERG channels, we created a homology model of hERG incorporating the region from the pore through to the end of the S6 helices of hERG, based on the KcsA crystal structure coordinates. Fig. 1B shows a schematic representation of the hERG pore regions that were modeled. Molecular dynamics simulations were then utilized to compare the conformational dynamics of the S6 helices of G648A, G657A, G648A/G657A, and WT hERG channels. Simulations were run for 20 ns, which is insufficient time to address gating processes as these occur on the millisecond to second time scale, but allows the native flexibility of S6 to be determined from the analysis of its mobility (e.g. (41)). The trajectories of S6 can be visualized by taking snapshots at equally spaced intervals and superimposing the N-terminal residues so that the bending of the helices is clearly presented (Fig. 5, A–D). Principal components analysis was used to analyze the swivel and kink movements of S6 during the simulations. Comparison of the different channels showed that the S6 kinking, observed in the WT channel, was also detected in the mutant channels. The analyses clearly demonstrate that there is flexibility in the vicinity of position 648, but also that mutation of the glycine to alanine does not significantly reduce the extent of the kinking or swiveling components of S6 motion. The mean kink angle of all four subunits in the simulations was 20.2°, 16.2°, 18.7°, and 17.2° for WT, G648A, G657A, and G648A/G657A hERG respectively. While the mean swivel angle was 46.2°, 42.9°, 27.9°, and 33.8° for WT, G648A, G657A, and G648A/G657A hERG, respectively. Although the helical kink is not as pronounced as the observed 25.7° from the KvAP crystal structure, it deviates significantly from the 7.9° of the initial structure. Moreover the swivel angles are closer to the open state of 43.5°, than the closed state 4.8°. These results suggest that there is inherent flexibility in the {alpha}-helix of hERG channels and this flexibility does not require glycine residues per se.


Figure 5
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  		FIGURE 5.
Molecular dynamics simulations illustrating S6 flexibility. Cartoon representations of the S6 helices. An average structure for each 20 ns simulation is shown in black, and compared with the initial closed inner-helix structure, based on KcsA (white) and the open (gray) S6 helix of KvAP. The simulations were done on the tetrameric pore domain, but for clarity only one S6/inner helix is shown. The location of the putative glycine gating hinge is marked by a star. A, WT; B, G648A; C, G657A; and D, G648A/G657A hERG. The definitions of the kink and swivel motions are illustrated by arrows in A. The simulations demonstrate that mutating the glycine residues to alanine has limited impact on the intrinsic flexibility of the hERG S6 helix.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we present evidence that it is not the flexibility provided by two glycines on the inner helix that is critical for hERG channel gating, but the close packing that is facilitated by the small side chain of this amino acid. Substitution of glycine for alanine, which normally stabilizes {alpha}-helices (22), has minor effects on activation gating and open channel block by terfenadine (24). Increasing the side chain volume at positions 648 and 657 facilitates channel opening rather than obstructing it. Molecular dynamics simulations indicate that there is little impact of alanine substitution on the kinking or swiveling motions of the S6 helices. We conclude that in hERG channels there is sufficient inherent flexibility of the inner helices that glycines are not required as hinge points for gating and may have a much greater importance for protein packing.

Our results on hERG are at variance with the findings of most studies on Kv, BK, and G protein-dependent K+ channels, which suggest the opening of the pore occurs by a bending of the inner helices at either a glycine gating hinge or at a Pro-X-Pro motif located seven residues downstream of the glycine hinge (7, 10, 17, 48, 49). The crystal structures of MthK (8) and KvAP (9) show a bent helix compared with the closed structures of KcsA (5) and this, together with the high degree of conservation of a glycine at this position in many potassium channels, led to the glycine hinge hypothesis (7). Close inspection of the Kv1.2 crystal structure reveals a definite kink and swivel at the glycine that is not readily apparent visually due to the more pronounced structural impact of the Pro-Val-Pro motif. Glycines permit a wide range of motion that is sterically impeded for all other amino acids. Functional studies in which the glycine hinge is mutated to a variety of different amino acids have usually resulted in loss of channel function (13, 14, 42, 49). This could be restored by inserting a glycine in neighboring positions (14) or substituting the glycine for a proline, which shifts the V0.5,act to negative potentials or renders the channel constitutively open (13). These studies supported the role of the conserved glycine as a gating hinge. However, not all channels behave in this fashion. Mutation of the putative glycine hinge to alanine in NaChBac produced only a –9 mV shift in the voltage dependence of activation, with very little effect on deactivation rates (23). Although the glycine hinge position is the most highly conserved residue on the inner helix, with 80% conservation (49), there are other K+ and CNG channels that lack glycines altogether in the homologous position. Clearly there are channels that may function without a glycine at the putative hinge position.

The Pro-X-Pro motif of S6 has been identified as a second key region for Kv channel gating. Proline residues may function as a point of weakness for bending during gating. The Pro-X-Pro motif has previously been proposed to insert a pronounced kink in the S6 helices (50, 51). The Kv1.2 crystal suggests that the first proline induces a ~30° kink in the S6 helix by breaking the hydrogen bonding network of the {alpha}-helix (10). Mutation of this motif has dramatic consequences for activation gating, with complete loss of channel function or large positive shifts in V0.5,act and a slowing of activation kinetics (19). In hERG, Gly657 is in alignment with the second Pro residue, and this glycine is also highly conserved in other EAG, Kir and hyperpolarization activated channel families. Although this more intracellular region of S6 could be a second site for bending, our results suggest neither glycine is absolutely required for activation gating. Experimental results and molecular dynamics simulations suggest the G648A/G657A mutant opens in a similar manner to the WT channel. Perhaps this is because hERG activation gating is conferred by a more extended length of S6. However, it is more likely that the flexibility required for channel opening can be provided by alanine and other amino acids. Molecular dynamics simulations of the channel pore indicate that bending of S6 occurs at position 648, with the C-terminal end of the pore helices having a crucial role as a pivot point. It is interesting to note that despite the curvature of the inner helix of open crystal structures, the phi and psi angles of the backbone do not differ dramatically from those of closed structures, with all residues lying in the {alpha}-helical region of the Ramachandran plot. Thus, the greater flexibility that glycines can provide is not required for the conformational changes during activation gating. A recent study by Rosenhouse-Dantsker and Logothetis on G protein-gated Kir3.4 channels came to a similar conclusion (52). Unlike hERG, most substitutions of Gly175 (equivalent to Gly648 in hERG) caused a decrease in Kir3.4 channel current. However, some mutants (e.g. G175A) produced substantial currents, arguing against the absolute requirement for a glycine hinge. Moreover, non-functional Gly175 mutants could be rescued by substitution of neighboring residues. Thus Gly (and some other amino acids) minimize local interactions that would otherwise hinder channel function.

The tight helix packing permitted by the glycine residues appears to be an important requirement at position 648 and 657. A fundamental difference between mutations of the proposed glycine gating hinge in Shaker and hERG is that Shaker mutations shift the gating equilibrium to the closed state (14, 17, 19), whereas hERG mutations shift it to the open state. Good correlations exist between side chain volume and hERG gating. The hERG mutations could function either to stabilize the open state or destabilize the closed state. At position 648, our results suggest that stabilization of the open state occurs because there is a substantial slowing of deactivation and the time constants correlate with side chain volume. It is interesting to note that other hERG mutations, such as D540K on the S4-S5 linker, destabilize the closed state and permit entry into the open state in response to either depolarization or hyperpolarization (25). Indeed, specific residues on both the S4-S5 linker and S6 of hERG are required to stabilize the closed state, and without them channels are constitutively open at negative potentials (53, 54). hERG is intrinsically more stable in the open state (the opposite to Shaker (17)), suggesting tangible differences in pore energetics. These findings also suggest that not only is the S4-S5 linker acting as a mechanical lever to open channels, but the voltage sensor also has to do positive work upon repolarization to close the channel. This could have important implications for the gating of hyperpolarization activated channels. S4-S5 linker mutations of HCN2 channels uncouple the voltage sensor from the pore, leaving it constitutively open (55). Thus, HCN2 channels, as with hERG, are more stable in the open state. Further work is needed to determine if the energetics of HCN pore opening (perhaps because of packing constraints in the pore) contribute to hyperpolarization-dependent gating.

In conclusion, mutation of S6 glycine residues fails to prevent hERG channel gating. It appears likely that these two glycine residues are important for gating due to their small side chain size permitting close packing rather than, as is widely believed, for their ability to introduce flexibility within {alpha}-helices. The bending of S6 with activation is likely to occur at a position close to 648. Our study provides further evidence that the role of highly conserved glycines in K+ channels may be to limit interactions with neighboring residues in closely packed and functionally important regions of the pore (52). However, hERG differs from other K+ channels in one crucial respect, because Gly substitutions increase open probability rather than limiting it.


   FOOTNOTES
 
* This work was supported in part by a career establishment award from the Medical Research Council (to J. S. M.), by a Pfizer-BBSRC CASE studentship (to S. D.), and a Novartis-MRC CASE studentship (to P. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S3. Back

1 To whom correspondence should be addressed: Dept. of Cell Physiology and Pharmacology, Medical Sciences Bldg., University of Leicester, University Road, Leicester LE1 9HN, UK. Tel.: 44-116-2928360; Fax: 44-116-252-5045; E-mail: jm109{at}le.ac.uk.

2 The abbreviations used are: Kv channels, voltage-gated family of potassium channels; hERG, human ether-à-go-go-related gene; POPC, palmitoyl-ole-oyl-phosphatidylcholine; MES, 4-morpholineethanesulfonic acid; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Seung Ho Kang and Madura Batu-wangala for technical support. We would also like to thank Alessandro Grottesi, Zara Sands, and Mark Sansom for their assistance with the molecular dynamics simulations.



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