Tryptophan scanning mutagenesis of the HERG K+ channel: the S4 domain is loosely packed and likely to be lipid exposed
1 Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia
2 Department of Medicine, UNSW, Victoria Street, Sydney, NSW 2010, Australia
Abstract
Inherited mutations or drug-induced block of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death. The fourth transmembrane domain (S4) of these channels contains multiple positive charges that move across the membrane electric field in response to changes in transmembrane voltage. In HERG K+ channels, the movement of the S4 domain across the transmembrane electric field is particularly slow. To examine the basis of the slow movement of the HERG S4 domain and specifically to probe the relationship between the S4 domain with the lipid bilayer and rest of the channel protein, we individually mutated each of the S4 amino acids in HERG (L524–L539) to tryptophan, and characterized the activation and deactivation properties of the mutant channels in Xenopus oocytes, using two-electrode voltage-clamp methods. Tryptophan has a large bulky hydrophobic sidechain and so should be tolerated at positions that interact with lipid, but not at positions involved in close protein–protein interactions. Significantly, we found that all S4 tryptophan mutants were functional. These data indicate that the S4 domain is loosely packed within the rest of the voltage sensor domain and is likely to be lipid exposed. Further, we identified residues K525, R528 and K538 as being the most important for slow activation of the channels.
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Introduction
Voltage-gated ion channels are crucial for a diverse range of physiological processes including neurotransmission, hormone secretion and regulation of the heart beat (Ashcroft, 2000). Voltage-gated sodium channels are responsible for the rapid upstroke of the action potential in atrial and ventricular myocytes, voltage-gated calcium channels are important for excitation–contraction coupling in cardiac myocytes and voltage-gated potassium (VGK) channels are critical for repolarization of the cardiac action potential (Noble, 1979). Furthermore, inherited mutations of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death (Keating & Sanguinetti, 2001; Subbiah et al. 2004a). HERG K+ channels are also the molecular target for many drugs that cause drug-induced cardiac arrhythmias (Sanguinetti & Bennett, 2003).
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VGK channels are composed of four subunits (MacKinnon, 1991), each with six transmembrane domains (S1–S6; see Fig. 1). Extensive experimental data supports the hypothesis that the highly positively charged S4 domain constitutes the principal voltage sensor for activation of these channels (reviewed in Bezanilla, 2000), including HERG K+ channels (Smith & Yellen, 2002; Piper et al. 2003). HERG channels have very atypical kinetics of slow activation and deactivation and rapid voltage-dependent inactivation (Smith et al. 1996; Spector et al. 1996; Schonherr & Heinemann, 1996; Wang et al. 1997; Zhou et al. 1998; Vandenberg et al. 2004). These characteristics, which are unique to HERG, are critical for the role the channel plays in cardiac repolarization (Smith et al. 1996; Spector et al. 1996; Viswanathan et al. 1999) as well as in protection against arrhythmias initiated by ectopic beats (Miller, 1996; Lu et al. 2001). Gating charge measurements (Piper et al. 2003) and measurements of fluorescent tags attached to the S3–S4 linker (Smith & Yellen, 2002) have shown that slow activation of HERG is due to slow movement of the S4 domain relative to the transmembrane electric field. Recent scanning mutagenesis studies of the HERG S4 domain (Subbiah et al. 2004b; Zhang et al. 2004; Piper et al. 2005) have shown that the first three charges in the HERG S4 domain, K525, R528 and R531, are the charges predominantly responsible for voltage sensing. However the reasons as to why the HERG voltage sensor residues move so slowly across the transmembrane electric field remain unknown.
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A, individual subunits contain 6 transmembrane domains (S1–S6). The S4 domain contains multiple positive charges and is the principal voltage sensor. The S5–S6 domains and an intervening P-loop form the pore region. B, the channel is a tetramer, with the ion conduction pathway formed by S5–S6 (grey) and surrounded by the voltage sensor domains (S1–S4). C, the voltage sensor regions of HERG (residues 517–542), KvAP (residues 111–137) and Kv1.2 (residues 262–314) were aligned with ClustalW (Thompson et al. 1994). Underlined regions represent S3b and S4 regions in KvAP. Residues highlighted in grey in HERG, were individually mutated to tryptophan in this study.
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Many models have been proposed for the mechanism by which the charged S4 domain moves across the transmembrane electric field including the sliding-helix (Guy & Seetharamulu, 1986; Keynes & Elinder, 1999), paddle (Jiang et al. 2003b, 2004; Long et al. 2005a, b) and transporter (Starace & Bezanilla, 2004; Bell et al. 2004; Posson et al. 2005; Chanda et al. 2005) models. These models make quite different predictions about the environment in which the S4 domain resides: surrounded by a proteinaceous jacket (Sliding-helix model), exposed to the lipid (paddle model) or adjacent to water filled crevices and loosely packed with S1–S3 domains (transporter model). The environment surrounding S4 is likely to have a significant impact on the rate at which the S4 domain can traverse the transmembrane electrical field. We therefore proceeded to probe the environment surrounding S4 in HERG K+ channels using tryptophan scanning mutagenesis. The rationale for this approach is that tryptophan has a bulky and hydrophobic sidechain and so should be tolerated in positions that interact with lipid or are involved in loose protein–protein interactions but less well tolerated, or not tolerated at all, in positions involved in tight protein–protein interactions (Choe et al. 1995; Collins et al. 1997). This strategy has been applied successfully to define the lipid–protein interface of transmembrane domains in many ion channels, including ROMK1 (Choe et al. 1995), Kir2.1 (Collins et al. 1997), and Shaker (Monks et al. 1999; Hong & Miller, 2000; Li-Smerin et al. 2000a; Hackos et al. 2002; Sukhareva et al. 2003), but not previously applied to the S4 domain of any voltage-gated ion channel.
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Significantly, we found that all S4 tryptophan mutants were functional, which indicates that the S4 domain is most likely lipid exposed. The data also indicate that S4 must be loosely packed and so are not consistent with close interactions between S4 and S3b. The data are therefore not fully consistent either with the traditional sliding helix or with the paddle models of voltage sensing. Lipid-exposure and loose packing of the S4 domain can be most easily interpreted on the basis of a recent hybrid model that is usually referred to as the transporter model (Starace & Bezanilla, 2004; Blaustein & Miller, 2004; Posson et al. 2005; Chanda et al. 2005).
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Methods
Molecular biology
HERG cDNA (a gift from Gail Robertson, University of Wisconsin) was subcloned into a pBluescript vector containing the 5' untranslated region and 3' untranslated region of the Xenopus laevis-globin gene (a gift from Robert Vandenberg, University of Sydney). Mutagenesis and cRNA synthesis were performed as previously described (Subbiah et al. 2004b). Mutant channels are referred to using the single letter code for WT residue, the residue number and then the single letter code of the mutation. For example, mutation of lysine 525 to a tryptophan is referred to as K525W. The sequences of S4 and all the mutants made in this study are shown in Fig. 1.
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Electrophysiology
Xenopus laevis oocytes were prepared as previously described (Clarke et al. 2000). Briefly, female Xenopus laevis frogs were anaesthetized in 0.17% w/v tricaine and segments of the ovarian lobes were removed through a small abdominal incision. The follicular layer was removed by digestion for 2–3 h with 2 mg ml–1 collagenase A (Boehringer Mannheim USA). Stage V and VI oocytes were isolated, stored in tissue culture dishes containing ND96 (containing, mM: 2.0 KCl, 96.0 NaCl, 1.8 CaCl2, 1.0 MgCl2, 5.0 Hepes, pH adjusted to 7.5 with NaOH), 2.5 mM pyruvic acid sodium salt and 0.5 mM theophylline supplemented with 10 μg ml–1 gentamicin, adjusted to pH 7.5 with NaOH and incubated at 18°C. Oocytes were injected with 40 nl of cRNA (1 ng nl–1) and incubated for 24–72 h. For electrophysiological recordings, oocytes were superfused with ND96 bath solution at room temperature (21–22°C). Frogs were humanely killed following final oocyte collection and all experiments were approved by the Animal Ethics Committee of the University of Sydney, NSW, Australia.
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The voltage dependence of current activation was assessed using standard tail current analysis (Wang et al. 1997; Subbiah et al. 2004b). Cells were depolarized to potentials in the range –110 mV to +90 mV (depending on the mutant being studied) and tail currents recorded at –70 mV, or at –150 mV if there was appreciable channel activation at potentials below –70 mV. For mutants with slow activation kinetics there will be a depolarizing shift in the voltage dependence of current activation if the depolarization steps are not of adequate length (Viloria et al. 2000). Therefore, 8 s depolarization steps were used to minimize this effect. Tail current data were normalized to the maximum current value (Imax) and fitted with a Boltzmann function:
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where I/Imax is the relative tail current, V0.5 is the half-activation voltage, Vt is the test potential, and k is the slope factor. Alternatively the data were fitted with the thermodynamic form of the Boltzmann function:
where, G0 is the work done at 0 mV, zg is the effective number of gating charges moving across the membrane electric field E, F is Faraday's constant, R is the universal gas constant and T is absolute temperature. Equations (1) and (2) are equivalent, but from eqn (2) one can calculate the effect of each mutation on changes in the chemical potential (G0) and electrostatic potential (–zgEF) that drive activation. Furthermore, the effect of mutations on the chemical potential energy of steady state activation could be calculated:
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It is well known that HERG K+ channels undergo transitions through multiple preopen closed states before opening (Wang et al. 1997). However, the above analysis relates only to the difference in energy between the group of closed states and the group of open states and so the values of G0 can only be used to estimate how specific mutants affect the relative stability of the group of open states relative to the group of closed states.
Rates of activation were measured using an envelope of tails protocol, as previously described (Subbiah et al. 2004b), in the range 0 to +160 mV. The activation of HERG K+ channels shows a sigmoidal time course, indicative of transitions through multiple preopen closed states before finally opening (Wang et al. 1997; Subbiah et al. 2004b). Therefore to obtain the rate constant of activation we used an equation of the form:
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where is the latency and is the time constant of the subsequent activation time course (Wang et al. 1997; Subbiah et al. 2004b).
To quantify the effect of each mutant on the rate of activation at 0 mV (or at equivalent electrochemical potentials, see online Supplemental material, Fig. S1), we calculated a perturbation energy:
where R is the universal gas constant, T is absolute temperature and mut and WT are time constants of activation for mutant and WT channels.
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Rates of deactivation were measured from tail currents recorded at voltages in the range from –60 to –190 mV (depending on the mutant studied) after a step to +40 mV for 500 ms to inactivate the channels. These data were fitted with a single exponential function of the form:
where is the time constant of deactivation, and A and C are constants. In some mutants deactivation appeared to follow a bi-exponential time course. However, at voltages negative to the activation threshold of the channels the slow component never represented more than 20% of the deactivation process and so in these cases we fitted only the first half of the deactivation process. To quantify the effects of tryptophan mutants on rates of deactivation we calculated a perturbation energy, Gdeact, using eqn (5) (see above).
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Correction for volume of sidechain
Tryptophan has the largest sidechain volume of the 20 naturally occurring amino acids (Creighton, 1993). The mutations made in the present study resulted in changes in sidechain volume that ranged from 15 3 (arginine to tryptophan) to 96 3 (alanine to tryptophan) with an average change in sidechain volume of 43.1 3. To investigate whether perturbations introduced by mutation to tryptophan were biased by the magnitude of the change in sidechain volume we calculated a corrected Gw0 using the equation, derived by Li-Smerin and colleagues (Li-Smerin et al. 2000a):
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where Volave is the average change in sidechain volume for the dataset used in this study and Vol is the change in sidechain volume for the specific mutant.
Statistics
All curve fitting was done with non-transformed data using the automated least squares fitting algorithm incorporated into the Clampfit 9 software (Axon Instruments, Union City, CA, USA). All data are presented as mean ±S.E.M. (n). Statistical comparisons (performed using ANOVA followed by Dunnett's method) were carried out using Microsoft Excel. A P-value of < 0.05 was considered significant.
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Results
S4 tryptophan mutants are all functional
In the absence of a crystal structure of the HERG K+ channel voltage sensor domain, we can only estimate the likely location of the S4 domain in HERG. Alignment of the S4 domain of HERG with that of KvAP and Kv1.2 (the two channels for which X-ray crystal structures are available) suggests that the first positive charge in the putative HERG S4 domain, K525, aligns with the second positively charged residue in KvAP, R132, and Kv1.2, R294 (see Fig. 1C). This is consistent with previous studies of charge neutralization mutants in HERG, which suggest that only the first three positively charged residues in HERG (K525, R528 and R531) contribute to charge transfer (Zhang et al. 2004) compared to the first four positively charged residues for Shaker-related channels (Aggarwal & MacKinnon, 1996; Seoh et al. 1996). Based on this analysis it is likely that the region from L524 to L539 constitutes the core of the S4 domain in HERG.
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Every individual mutation of residues L524 to L539 in HERG to tryptophan was tolerated and had functional characteristics similar to wild-type (WT) HERG K+ channels (see Fig. 2). There was relatively little current at depolarized potentials as the channels open and inactivate, but much larger currents at –120 mV, despite the smaller electrochemical driving force for K+ (EK–95 mV), as the channels rapidly recover from inactivation but only deactivate slowly.
Typical examples of currents recorded from WT and S4 tryptophan mutants during depolarizing steps to +40 mV from a holding potential of –90 mV and then a repolarization step to –120 mV. All mutants show classical behaviour of HERG K+ channels with small current during depolarizing steps and hooked tail currents due to rapid recovery from inactivation and subsequent deactivation. Horizontal lines to left of each current trace indicate zero current level.
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Effects of tryptophan mutants on steady-state activation
To quantify the effect of each mutant on the energetics of channel activation, we fitted a Boltzmann function to the isochronal activation curves (see Fig. 3A and Table 1). From these fits we calculated, for each mutant, the perturbation to the difference in free energy between the closed and open states at 0 mV, i.e. G0 (see Methods). Five of the mutants (K525W, A527W, R528W, V533W and R534W), three involving positively charged residues, caused minimal perturbation to G0 (see Fig. 3B). Four mutants caused perturbations of > ± 3 kcal mol–1 (L524W, T526W, V535W and A536W). The remaining mutants caused perturbations ranging from ± 1 to ± 3 kcal mol–1, where a negative value indicates stabilization of the group of open states relative to the group of closed states and a positive value indicates stabilization of the group of closed states relative to the group of open states at 0 mV.
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A, typical family of tail currents recorded from WT, A536W and V535W channels during isochronal activation voltage protocols (see inset). Traces marked with an arrow in each panel indicate the test voltage at which each channel was approximately 50% activated. B, amplitudes of the tail currents for WT (), A536W () and V535W () channels were normalized to the maximum current value (Imax). Data points shown represent averages (±S.E.M.) and the continuous lines represent the best fit of a Boltzmann function (see Methods). The values for G0, the work done at 0 mV, and zg, the effective number of gating charges moving across the membrane electric field, derived from the Boltzmann fits to these data are summarized in Table 1. C, bar graph showing G0 (i.e. G0=G0 (mutant) –G0 (WT)) for each mutant. Five mutants (K525W, A527W, R528W, V533W and R534W) caused minimal change in G0 (defined as G0 < 1 kcal mol–1, dashed line).
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It has been suggested that mutation of residues to tryptophan can introduce bias due to the large variations in the changes in side chain volume (Li-Smerin et al. 2000a), which does not appear to be the case here. The pattern of perturbation to the gating behaviour of HERG S4 tryptophan mutants was similar before and after correcting G0 values for changes in volume (see Table 1). The residues in which there was the largest change in |G0| when values have been corrected for change in size are the arginine residues. This is because there are relatively small changes in sidechain volume when mutating an arginine to a tryptophan (see eqn (7)).
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Effects of tryptophan mutants on rates of activation
Typical examples of currents recorded during protocols to measure rates of activation at 0 mV for WT, T526W and R531W channels are shown in Fig. 4A. T526W has a faster rate of activation at 0 mV whereas R531W has a slower rate of activation at 0 mV compared to WT channels. The majority of S4 tryptophan mutants had smaller time constants of activation (see Fig. 4B), and hence, faster rates of activation than WT channels. In particular, mutation of either of the lysine residues to tryptophan (K525W, K538W) caused marked acceleration of the rates of activation at 0 mV (see Fig. 4B).
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A, examples of rates of activation at 0 mV, measured using an envelope-of-tails protocol (see inset), for WT, T526W and R531W channels. The time constant of activation was obtained by fitting the envelope of peak tail currents with eqn (4) (see Methods). B, summary (mean ±S.E.M.) of time constants of activation for all S4 tryptophan mutants.
One concern with comparing the rates of activation at 0 mV, is that due to shifts in the voltage dependence of steady-state activation (see Fig. 3) the electrochemical potential for activation (chemical, G0, plus electrostatic, –zgEF) at any given voltage will be different for each mutant. We therefore measured the rates of activation for all the S4 tryptophan mutants and WT channels at multiple voltages in the range +40 to +160 mV and plotted the time constants of activation against the total electrochemical potential (–(G0–zgEF); see Fig. 5A. From these data we interpolated a value for the time constant of activation for each mutant channel at an electrochemical potential of 7 kcal mol–1 (dashed line, Fig. 5A and see Supplemental material Fig. S1 and Table S1) and then calculated a value for G of activation (see Fig. 5B; Table 2). We chose 7 kcal mol–1, as this was a value at which all channels would be fully activated and where we had data for all mutants. At an electrochemical potential of 7 kcal mol–1, the values of Gact showed a periodicity with the 1st, 3rd, 4th, 7th, 10th, 11th and 14th residues having either a positive or very small value for G. This pattern of perturbation to Gact was not altered when the values for Gact were corrected for changes in sidechain volume (see Table 2).
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A, time constants of activation for WT and S4 tryptophan mutant channels were obtained at voltages in the range 0 to +160 mV, and are plotted versus the total electrochemical potential (see Methods), i.e. –(G0–zgEF). Error bars are omitted for purposes of clarity. The mean data (±S.E.M.) for the interpolated time constants of activation calculated for a driving force at 7 kcal mol–1 for each mutant in at least n= 4 separate oocytes is shown in Supplemental material Table S1. B, plot of G. At an equivalent electrochemical potential of 7 kcal mol–1, there is a periodicity in the perturbation of G with the 1st, 3rd, 4th, 7th, 10th, 11th and 14th residues having either a positive or very small value for G.
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Effects of tryptophan mutants on rates of deactivation
Typical examples of tail currents recorded at –80 mV for WT, L539W and V535W channels are shown in Fig. 6A. Current traces have been normalized to the peak tail current to facilitate comparison of the rates of deactivation. In the traces shown in Fig. 6A, L539W channels deactivate more slowly than WT, whereas V535W channels deactivate more rapidly. The rates of deactivation at –80 mV for all S4 tryptophan mutants varied from 35 ± 2 ms for V535W to 1093 ± 6 ms for R528W compared to 198 ± 1 ms for WT HERG (see Fig. 6B). The problems with comparing rates of activation between mutant channels at a given voltage (see above) also applies to comparing rates of deactivation at a given voltage. We therefore measured rates of deactivation at multiple voltages in the range –60 to –190 mV and have plotted the rates of deactivation against the electrochemical potential for deactivation (see Fig. 7A). To quantify the effect of each mutant on the rate of deactivation at an electrochemical potential of 7 kcal mol–1, we calculated a perturbation energy Gdeact (eqn (5)) for each mutant relative to WT (see Fig. 7B; Table 2). There is a clear periodicity of perturbation to Gdeact at an electrochemical potential of 7 kcal mol–1 with the 1st, 2nd and then the 6th, 9th, 12th, 13th and 15th residues showing significantly more negative Gdeact values than the other residues (see Fig. 7D).
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A, typical examples of tail currents recorded at –80 mV for WT, L539W and V535W following a step to +40 mV to activate the channels (voltage protocol shown in inset). Traces have been normalized to the peak tail current to facilitate comparison of the rates of deactivation. In the traces shown, the currents for L539W decay more slowly than for WT, which in turn is slower than for V535W. B, summary (mean ±S.E.M.) of time constants of deactivation for all S4 tryptophan mutants at –80 mV.
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A, time constants of deactivation for WT and S4 tryptophan mutant channels were obtained at voltages in the range 0 to –190 mV, and are plotted versus the electrochemical potential, i.e. –(G0–zgEF). B, perturbation of deactivation, G, calculated using eqn (5) (see Methods), for time constants of deactivation calculated at an equivalent electrochemical potential of 7 kcal mol–1.
Are charged residues in the S4 of HERG exposed to a lipid or aqueous environment
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Many previous studies have suggested that the charged residues in S4 interact with an aqueous cavity (see, e.g. Gandhi et al. 2003; Laine et al. 2003; Ahern & Horn, 2004; Bell et al. 2004; Starace & Bezanilla, 2004; Tombola et al. 2005). Conversely, the crystal structures of KvAP and Kv1.2 suggested that some of the positively charged residues may interact with lipid (Jiang et al. 2003a; Long et al. 2005a). Our data, showing that tryptophan mutants of S4 charged residues are all tolerated, suggest that in HERG K+ channels some of the charged residues may be exposed to lipid in at least one conformational state of the channel. In previous studies we mutated each of the charged residues in the S4 domain of HERG (K525, R528, R531, R534, R537) to glutamine (Subbiah et al. 2004b) and we have extended this to K538 (see Fig. S2, Supplemental material). In all instances the glutamine substitution caused a similar or greater perturbation to G0 than a tryptophan substitution (see Fig. 8). The most significant differences between the tryptophan and glutamine substitutions occurred for R528 and R531 (see Supplemental material Table S1). Taken together, the data from the glutamine and tryptophan mutants suggest that the charged residues in the S4 domain (and in particular R528 and R531) are likely to interact with a lipid environment in one or more conformational states of the channel and so more readily permit substitution with the bulky hydrophobic tryptophan sidechain than glutamine.
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Comparison of perturbation to G0 for tryptophan (filled bars) and glutamine (open bars) mutants of the charged residues in the S4 domain of HERG. Glutamine substitution compared to tryptophan substitution of S4 charged residues caused a similar or greater perturbation to G0. The most significant differences observed between the tryptophan and glutamine substitutions were at R528 and R531.
Discussion
HERG K+ channels exhibit unusual kinetics characterized by slow activation and deactivation compared to rapid inactivation and recovery from inactivation. Previous studies have shown that slow activation is caused by slow movement of the S4 domain (Smith & Yellen, 2002; Piper et al. 2003). The mechanisms by which the S4 domain moves slowly, however, remain to be determined. In this study we have shown that every residue in the HERG S4 domain (from L524 to L539) can be mutated to tryptophan and still produce functional channels. In previous studies where tryptophan-scanning mutagenesis was employed in inward rectifier K+ channels or the pore of the Shaker K+ channel, between 15 and 55% of residues were intolerant of tryptophan substitution (Choe et al. 1995; Collins et al. 1997; Hackos et al. 2002; Sukharev et al. 2003). From these data, it was concluded that the non-functional tryptophan mutants identified residues involved in tight protein–protein interactions, whereas the remaining residues probably faced the lipid. In marked contrast to these studies, only 2 out of 67 residues in the S1, S2 and S3 domains of the Shaker K+ channel did not tolerate substitution to tryptophan (Monks et al. 1999; Hong & Miller, 2000). Based on these data Miller and colleagues concluded that the S1–S3 segments had a peripheral location, adjacent to lipid, and presumably surrounded and protected the S4 domain from the lipid. However, there will only be a periodicity in response to mutant scans if there are two environments that a domain is exposed to and the mutant residue perturbs interactions with one environment more than the other. Presumably, the two environments that S4 is exposed to are loose protein–protein interactions and a lipid environment. Therefore the observation that the S1–S3 segments can tolerate tryptophan substitutions at 65/67 locations indicates that these domains are adjacent to lipid and only loosely packed. By the same reasoning, we would have to conclude based on our experimental data for S4 tryptophan mutants of HERG that part the S4 domain in HERG is loosely packed within the voltage sensor and probably also faces the lipid. However, based on our data it is not possible to determine which residues of the HERG S4 domain face lipid. Furthermore, residues that interact with lipid may vary between different conformational states of the channel. Our observation that there was a helical periodicity to the effects of Tryptophan substitutions (see Figs 5B and 7B) is also consistent with previous studies indicating that the S4 domain must be -helical (Mulvey et al. 1989; Li-Smerin et al. 2000b; Jiang et al. 2003a; Long et al. 2005a).
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Our conclusion that the S4 domain must be loosely packed within the voltage sensor is somewhat at odds with the crystal structures of KvAP (Jiang et al. 2003a) and Kv1.2 (Long et al. 2005a), which indicate that the S4 and S3 domains are closely packed. However, given the relatively poor sequence homology between the S3b segment of HERG and KvAP (see Fig. 1C), one must be cautious with making structural predictions for HERG based on the KvAP and Kv1.2 voltage sensor structures.
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Slow activation of HERG
The slow activation of HERG is due to slow movement of the S4 domain (Smith & Yellen, 2002; Piper et al. 2003). This indicates that there must be a large energy barrier between the closed state and intermediate transition state. Mutants could therefore affect rates of activation either by stabilizing or destabilizing the closed or open states relative to each other or by destabilizing these states relative to an intermediate transition state.
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The residues that when mutated to tryptophan caused the largest acceleration in the rates of activation (after correcting for shifts in the voltage sensitivity of different mutants and alterations in sidechain volume; see column 3, Table 2) were K525, R528, R531, K538 and L539. In the case of K525, K538 and L539 these residues when mutated to tryptophan also showed marked acceleration of deactivation. Thus, one possibility is that mutation of K525, K538 and L539 results in stabilization of a transition state (and hence lower energy barrier) between the grouped closed and open states and so permit faster activation and faster deactivation. Further, K525W had minimal effect on the overall voltage dependence of the closed–open state equilibrium as measured by the Boltzmann distribution (see Fig. 4C). Thus in the case of K525W the effect of the mutation can be largely explained by stabilization of a transition state. K538W and L539W caused a modest destabilization of the closed state relative to the open state (negative G0, see Fig. 4C). Thus, in the case of K538 and L539, as well as stabilizing a transition state (see above) they must also be affecting the stability of the closed and/or open state.
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Mutation of R528 and R531 resulted in faster activation but no change or a slowing of deactivation (see Fig. 7). These residues correspond to R300 and R303 in Kv1.2 (see Fig. 1C) the residues that were shown to form salt bridges in the presumed open conformation of the Kv1.2 channel (Long et al. 2005a). If similar salt bridges exist in the open state of HERG then the R528 and R531 mutants would be predicted to destabilize the open state. Consistent with this R531W resulted in a significant destabilization of the open state relative to the closed state, as reflected in the Boltzmann distribution analysis of isochronal activation curves (see Fig. 4, Table 1). However, R528W did not. If the salt bridges were present in the transition state then one would expect the tryptophan mutants to destabilize the transition state and so slow activation (not observed) and deactivation (observed with R528W but not R531W). If the salt bridges are present in the closed state then one might expect tryptophan mutants to increase the rate of activation (observed with both R528W and R531W). Thus our data would be consistent with a salt bridge involving R528 present in at least one closed state and a transition state and a salt bridge involving R531 present in at least one closed state and one open state. It should also be noted that mutation of R531 or R528 to glutamine caused a greater perturbation to the overall open–closed state equilibrium than did mutation to tryptophan (see Fig. 7). These data suggest that in one or more conformational states R531 and R528 are likely to be exposed to lipid (although it is not necessarily the same conformational state for both residues). Overall, our data in conjunction with the data in the literature, are consistent with a model in which R528 and R531 participate in salt bridges in one or more but not all conformational states of the channel and are exposed to lipid in other conformational states of the channel.
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Scanning mutagenesis studies of the HERG S4 domain with glutamine identified K525Q, R528Q, R534Q (Subbiah et al. 2004b) and K538Q (see Supplemental material) as having significantly faster rates of activation than WT channels. In previous cysteine scanning (Zhang et al. 2004) and alanine scanning (Piper et al. 2005) mutagenesis studies of the HERG S4 domain, the rates of activation were not measured, so it is not possible to make direct comparisons with this study. However, in both the cysteine scanning and alanine scanning studies, mutations of K538 significantly destabilized the closed state of the channel, which would be consistent with faster rates of activation. Mutation of K525 also destabilized the closed state when mutated to cysteine (Subbiah et al. 2004b; Zhang et al. 2004) or was non-functional when mutated to alanine (Piper et al. 2005). The results for residues in the middle of S4 are more variable between the four studies. This suggests that the specific chemical properties of residues in the middle of S4 influence the activity of the channel. This in turn could be most easily explained by these residues contributing to specific protein–protein interactions within the voltage sensor domain, such as the putative salt bridges discussed above for R528 and R531.
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Together, the results from the four scanning mutagenesis studies suggest that the lysine residues at each end of the HERG S4 domain (K525 and K538) are important for stabilizing the closed state and thereby contributing to the slow activation of the channels. It is likely that residues in the middle of S4 may also be important for slow activation, with residues R528 and R531 key candidates for binding to residues in other transmembrane domains.
Slow deactivation of HERG
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The mutations that most perturbed the rates of deactivation (after correction for changes in electrochemical driving force and sidechain volume) were similar to those perturbing the rates of activation (see Supplemental material Table S1). In particular, mutating K525, R528 and K538 significantly affected rates of both activation and deactivation (as discussed above). There are however, some differences, e.g. mutating L524 and L532 had significant effects on deactivation but not activation and mutation of R531 had significant effects on activation but not deactivation. This is probably not surprising given that activation and deactivation are multistep processes and the measured rates of activation and deactivation are not necessarily measurements of the forward and reverse rates of the same step in the process. Thus, if mutation of L524 and L532 to tryptophan stabilizes the transition state for the rate-limiting step in the deactivation process but this same step was not rate limiting for activation, then the mutants would lead to faster deactivation but have minimal effect on activation.
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Models of voltage sensing
The conundrum of how charges can move across the transmembrane electric field, yet be shielded from the thermodynamic cost of being exposed to a hydrophobic lipid environment has puzzled biophysicists for many decades (Bezanilla, 2000). The sliding-helix model, in which it was proposed that the positively charged residues in S4 were cocooned in a proteinaceous jacket composed of the S1–S3 domains to protect them from the hydrophobic membrane interior, provided an elegant explanation for this conundrum (see, e.g. Keynes & Elinder (1999) for review). With minor modifications, the sliding-helix model has remained the accepted paradigm for the mechanism of voltage sensing, until very recently. However, data from the crystal structure of KvAP (Jiang et al. 2003a), supported by evidence from biotin–avidin accessibility studies (Jiang et al. 2003b) and electron microscopy studies (Jiang et al. 2004) of the KvAP channel, are incompatible with the sliding-helix model. MacKinnon and colleagues postulated an alternative model, the paddle model, in which the S4 domain lies at the periphery of the channel and is able to move freely across the lipid membrane in response to changes in membrane voltage (Jiang et al. 2003b). However, all the evidence, to date, in support of the hydrophobic cation model of voltage sensing has come from studies of the bacterial KvAP channel. Could it also explain voltage sensing in mammalian voltage-gated ion channelsThis is of particular relevance to cardiac biology as voltage-gated ion channels are central to the regulation of the heart beat. Furthermore, mutations in the voltage sensor domains of voltage-gated ion channels can predispose to an increased risk of cardiac arrhythmias (Clancy & Kass, 2005).
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Our data showing that every residue in the S4 domain of HERG can tolerate a tryptophan substitution places significant constraints on the orientation of S4 with respect to the remainder of the channel and the lipid bilayer. Our data suggest that the S4 domain must be loosely packed with respect to the rest of the channel and at least partially lipid exposed. The tolerance of tryptophan substitutions is consistent with a peripheral location for S4 as suggested in the paddle model proposed by MacKinnon and colleagues (Jiang et al. 2003b, 2004; Long et al. 2005b). However, it is not consistent with the traditional sliding-helix model where S4 is cocooned within a proteinaceous jacket composed of the S1–S3 domains (Keynes & Elinder, 1999). The tolerance of tryptophan substitutions, however, does not support a tight interaction between S4 and S3 as suggested in the paddle model (Jiang et al. 2003b; Long et al. 2005a).
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Recently, Starace & Bezanilla (2004) and Tombola et al. (2005) have suggested a modification to the sliding-helix model, known as the transporter model, in which the S1–S3 domains are loosely packed around S4 and changes in the transmembrane electric field, consequent to movement of S1–S3 relative to S4, result in the effective movement of S4 charges across the membrane. The tolerance of tryptophan substitutions at all residues in the S4 domain of HERG is consistent with loose packing of the S4 domain as proposed in this transporter model.
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2 Department of Medicine, UNSW, Victoria Street, Sydney, NSW 2010, Australia
Abstract
Inherited mutations or drug-induced block of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death. The fourth transmembrane domain (S4) of these channels contains multiple positive charges that move across the membrane electric field in response to changes in transmembrane voltage. In HERG K+ channels, the movement of the S4 domain across the transmembrane electric field is particularly slow. To examine the basis of the slow movement of the HERG S4 domain and specifically to probe the relationship between the S4 domain with the lipid bilayer and rest of the channel protein, we individually mutated each of the S4 amino acids in HERG (L524–L539) to tryptophan, and characterized the activation and deactivation properties of the mutant channels in Xenopus oocytes, using two-electrode voltage-clamp methods. Tryptophan has a large bulky hydrophobic sidechain and so should be tolerated at positions that interact with lipid, but not at positions involved in close protein–protein interactions. Significantly, we found that all S4 tryptophan mutants were functional. These data indicate that the S4 domain is loosely packed within the rest of the voltage sensor domain and is likely to be lipid exposed. Further, we identified residues K525, R528 and K538 as being the most important for slow activation of the channels.
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Introduction
Voltage-gated ion channels are crucial for a diverse range of physiological processes including neurotransmission, hormone secretion and regulation of the heart beat (Ashcroft, 2000). Voltage-gated sodium channels are responsible for the rapid upstroke of the action potential in atrial and ventricular myocytes, voltage-gated calcium channels are important for excitation–contraction coupling in cardiac myocytes and voltage-gated potassium (VGK) channels are critical for repolarization of the cardiac action potential (Noble, 1979). Furthermore, inherited mutations of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death (Keating & Sanguinetti, 2001; Subbiah et al. 2004a). HERG K+ channels are also the molecular target for many drugs that cause drug-induced cardiac arrhythmias (Sanguinetti & Bennett, 2003).
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VGK channels are composed of four subunits (MacKinnon, 1991), each with six transmembrane domains (S1–S6; see Fig. 1). Extensive experimental data supports the hypothesis that the highly positively charged S4 domain constitutes the principal voltage sensor for activation of these channels (reviewed in Bezanilla, 2000), including HERG K+ channels (Smith & Yellen, 2002; Piper et al. 2003). HERG channels have very atypical kinetics of slow activation and deactivation and rapid voltage-dependent inactivation (Smith et al. 1996; Spector et al. 1996; Schonherr & Heinemann, 1996; Wang et al. 1997; Zhou et al. 1998; Vandenberg et al. 2004). These characteristics, which are unique to HERG, are critical for the role the channel plays in cardiac repolarization (Smith et al. 1996; Spector et al. 1996; Viswanathan et al. 1999) as well as in protection against arrhythmias initiated by ectopic beats (Miller, 1996; Lu et al. 2001). Gating charge measurements (Piper et al. 2003) and measurements of fluorescent tags attached to the S3–S4 linker (Smith & Yellen, 2002) have shown that slow activation of HERG is due to slow movement of the S4 domain relative to the transmembrane electric field. Recent scanning mutagenesis studies of the HERG S4 domain (Subbiah et al. 2004b; Zhang et al. 2004; Piper et al. 2005) have shown that the first three charges in the HERG S4 domain, K525, R528 and R531, are the charges predominantly responsible for voltage sensing. However the reasons as to why the HERG voltage sensor residues move so slowly across the transmembrane electric field remain unknown.
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A, individual subunits contain 6 transmembrane domains (S1–S6). The S4 domain contains multiple positive charges and is the principal voltage sensor. The S5–S6 domains and an intervening P-loop form the pore region. B, the channel is a tetramer, with the ion conduction pathway formed by S5–S6 (grey) and surrounded by the voltage sensor domains (S1–S4). C, the voltage sensor regions of HERG (residues 517–542), KvAP (residues 111–137) and Kv1.2 (residues 262–314) were aligned with ClustalW (Thompson et al. 1994). Underlined regions represent S3b and S4 regions in KvAP. Residues highlighted in grey in HERG, were individually mutated to tryptophan in this study.
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Many models have been proposed for the mechanism by which the charged S4 domain moves across the transmembrane electric field including the sliding-helix (Guy & Seetharamulu, 1986; Keynes & Elinder, 1999), paddle (Jiang et al. 2003b, 2004; Long et al. 2005a, b) and transporter (Starace & Bezanilla, 2004; Bell et al. 2004; Posson et al. 2005; Chanda et al. 2005) models. These models make quite different predictions about the environment in which the S4 domain resides: surrounded by a proteinaceous jacket (Sliding-helix model), exposed to the lipid (paddle model) or adjacent to water filled crevices and loosely packed with S1–S3 domains (transporter model). The environment surrounding S4 is likely to have a significant impact on the rate at which the S4 domain can traverse the transmembrane electrical field. We therefore proceeded to probe the environment surrounding S4 in HERG K+ channels using tryptophan scanning mutagenesis. The rationale for this approach is that tryptophan has a bulky and hydrophobic sidechain and so should be tolerated in positions that interact with lipid or are involved in loose protein–protein interactions but less well tolerated, or not tolerated at all, in positions involved in tight protein–protein interactions (Choe et al. 1995; Collins et al. 1997). This strategy has been applied successfully to define the lipid–protein interface of transmembrane domains in many ion channels, including ROMK1 (Choe et al. 1995), Kir2.1 (Collins et al. 1997), and Shaker (Monks et al. 1999; Hong & Miller, 2000; Li-Smerin et al. 2000a; Hackos et al. 2002; Sukhareva et al. 2003), but not previously applied to the S4 domain of any voltage-gated ion channel.
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Significantly, we found that all S4 tryptophan mutants were functional, which indicates that the S4 domain is most likely lipid exposed. The data also indicate that S4 must be loosely packed and so are not consistent with close interactions between S4 and S3b. The data are therefore not fully consistent either with the traditional sliding helix or with the paddle models of voltage sensing. Lipid-exposure and loose packing of the S4 domain can be most easily interpreted on the basis of a recent hybrid model that is usually referred to as the transporter model (Starace & Bezanilla, 2004; Blaustein & Miller, 2004; Posson et al. 2005; Chanda et al. 2005).
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Methods
Molecular biology
HERG cDNA (a gift from Gail Robertson, University of Wisconsin) was subcloned into a pBluescript vector containing the 5' untranslated region and 3' untranslated region of the Xenopus laevis-globin gene (a gift from Robert Vandenberg, University of Sydney). Mutagenesis and cRNA synthesis were performed as previously described (Subbiah et al. 2004b). Mutant channels are referred to using the single letter code for WT residue, the residue number and then the single letter code of the mutation. For example, mutation of lysine 525 to a tryptophan is referred to as K525W. The sequences of S4 and all the mutants made in this study are shown in Fig. 1.
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Electrophysiology
Xenopus laevis oocytes were prepared as previously described (Clarke et al. 2000). Briefly, female Xenopus laevis frogs were anaesthetized in 0.17% w/v tricaine and segments of the ovarian lobes were removed through a small abdominal incision. The follicular layer was removed by digestion for 2–3 h with 2 mg ml–1 collagenase A (Boehringer Mannheim USA). Stage V and VI oocytes were isolated, stored in tissue culture dishes containing ND96 (containing, mM: 2.0 KCl, 96.0 NaCl, 1.8 CaCl2, 1.0 MgCl2, 5.0 Hepes, pH adjusted to 7.5 with NaOH), 2.5 mM pyruvic acid sodium salt and 0.5 mM theophylline supplemented with 10 μg ml–1 gentamicin, adjusted to pH 7.5 with NaOH and incubated at 18°C. Oocytes were injected with 40 nl of cRNA (1 ng nl–1) and incubated for 24–72 h. For electrophysiological recordings, oocytes were superfused with ND96 bath solution at room temperature (21–22°C). Frogs were humanely killed following final oocyte collection and all experiments were approved by the Animal Ethics Committee of the University of Sydney, NSW, Australia.
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The voltage dependence of current activation was assessed using standard tail current analysis (Wang et al. 1997; Subbiah et al. 2004b). Cells were depolarized to potentials in the range –110 mV to +90 mV (depending on the mutant being studied) and tail currents recorded at –70 mV, or at –150 mV if there was appreciable channel activation at potentials below –70 mV. For mutants with slow activation kinetics there will be a depolarizing shift in the voltage dependence of current activation if the depolarization steps are not of adequate length (Viloria et al. 2000). Therefore, 8 s depolarization steps were used to minimize this effect. Tail current data were normalized to the maximum current value (Imax) and fitted with a Boltzmann function:
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where I/Imax is the relative tail current, V0.5 is the half-activation voltage, Vt is the test potential, and k is the slope factor. Alternatively the data were fitted with the thermodynamic form of the Boltzmann function:
where, G0 is the work done at 0 mV, zg is the effective number of gating charges moving across the membrane electric field E, F is Faraday's constant, R is the universal gas constant and T is absolute temperature. Equations (1) and (2) are equivalent, but from eqn (2) one can calculate the effect of each mutation on changes in the chemical potential (G0) and electrostatic potential (–zgEF) that drive activation. Furthermore, the effect of mutations on the chemical potential energy of steady state activation could be calculated:
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It is well known that HERG K+ channels undergo transitions through multiple preopen closed states before opening (Wang et al. 1997). However, the above analysis relates only to the difference in energy between the group of closed states and the group of open states and so the values of G0 can only be used to estimate how specific mutants affect the relative stability of the group of open states relative to the group of closed states.
Rates of activation were measured using an envelope of tails protocol, as previously described (Subbiah et al. 2004b), in the range 0 to +160 mV. The activation of HERG K+ channels shows a sigmoidal time course, indicative of transitions through multiple preopen closed states before finally opening (Wang et al. 1997; Subbiah et al. 2004b). Therefore to obtain the rate constant of activation we used an equation of the form:
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where is the latency and is the time constant of the subsequent activation time course (Wang et al. 1997; Subbiah et al. 2004b).
To quantify the effect of each mutant on the rate of activation at 0 mV (or at equivalent electrochemical potentials, see online Supplemental material, Fig. S1), we calculated a perturbation energy:
where R is the universal gas constant, T is absolute temperature and mut and WT are time constants of activation for mutant and WT channels.
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Rates of deactivation were measured from tail currents recorded at voltages in the range from –60 to –190 mV (depending on the mutant studied) after a step to +40 mV for 500 ms to inactivate the channels. These data were fitted with a single exponential function of the form:
where is the time constant of deactivation, and A and C are constants. In some mutants deactivation appeared to follow a bi-exponential time course. However, at voltages negative to the activation threshold of the channels the slow component never represented more than 20% of the deactivation process and so in these cases we fitted only the first half of the deactivation process. To quantify the effects of tryptophan mutants on rates of deactivation we calculated a perturbation energy, Gdeact, using eqn (5) (see above).
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Correction for volume of sidechain
Tryptophan has the largest sidechain volume of the 20 naturally occurring amino acids (Creighton, 1993). The mutations made in the present study resulted in changes in sidechain volume that ranged from 15 3 (arginine to tryptophan) to 96 3 (alanine to tryptophan) with an average change in sidechain volume of 43.1 3. To investigate whether perturbations introduced by mutation to tryptophan were biased by the magnitude of the change in sidechain volume we calculated a corrected Gw0 using the equation, derived by Li-Smerin and colleagues (Li-Smerin et al. 2000a):
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where Volave is the average change in sidechain volume for the dataset used in this study and Vol is the change in sidechain volume for the specific mutant.
Statistics
All curve fitting was done with non-transformed data using the automated least squares fitting algorithm incorporated into the Clampfit 9 software (Axon Instruments, Union City, CA, USA). All data are presented as mean ±S.E.M. (n). Statistical comparisons (performed using ANOVA followed by Dunnett's method) were carried out using Microsoft Excel. A P-value of < 0.05 was considered significant.
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Results
S4 tryptophan mutants are all functional
In the absence of a crystal structure of the HERG K+ channel voltage sensor domain, we can only estimate the likely location of the S4 domain in HERG. Alignment of the S4 domain of HERG with that of KvAP and Kv1.2 (the two channels for which X-ray crystal structures are available) suggests that the first positive charge in the putative HERG S4 domain, K525, aligns with the second positively charged residue in KvAP, R132, and Kv1.2, R294 (see Fig. 1C). This is consistent with previous studies of charge neutralization mutants in HERG, which suggest that only the first three positively charged residues in HERG (K525, R528 and R531) contribute to charge transfer (Zhang et al. 2004) compared to the first four positively charged residues for Shaker-related channels (Aggarwal & MacKinnon, 1996; Seoh et al. 1996). Based on this analysis it is likely that the region from L524 to L539 constitutes the core of the S4 domain in HERG.
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Every individual mutation of residues L524 to L539 in HERG to tryptophan was tolerated and had functional characteristics similar to wild-type (WT) HERG K+ channels (see Fig. 2). There was relatively little current at depolarized potentials as the channels open and inactivate, but much larger currents at –120 mV, despite the smaller electrochemical driving force for K+ (EK–95 mV), as the channels rapidly recover from inactivation but only deactivate slowly.
Typical examples of currents recorded from WT and S4 tryptophan mutants during depolarizing steps to +40 mV from a holding potential of –90 mV and then a repolarization step to –120 mV. All mutants show classical behaviour of HERG K+ channels with small current during depolarizing steps and hooked tail currents due to rapid recovery from inactivation and subsequent deactivation. Horizontal lines to left of each current trace indicate zero current level.
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Effects of tryptophan mutants on steady-state activation
To quantify the effect of each mutant on the energetics of channel activation, we fitted a Boltzmann function to the isochronal activation curves (see Fig. 3A and Table 1). From these fits we calculated, for each mutant, the perturbation to the difference in free energy between the closed and open states at 0 mV, i.e. G0 (see Methods). Five of the mutants (K525W, A527W, R528W, V533W and R534W), three involving positively charged residues, caused minimal perturbation to G0 (see Fig. 3B). Four mutants caused perturbations of > ± 3 kcal mol–1 (L524W, T526W, V535W and A536W). The remaining mutants caused perturbations ranging from ± 1 to ± 3 kcal mol–1, where a negative value indicates stabilization of the group of open states relative to the group of closed states and a positive value indicates stabilization of the group of closed states relative to the group of open states at 0 mV.
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A, typical family of tail currents recorded from WT, A536W and V535W channels during isochronal activation voltage protocols (see inset). Traces marked with an arrow in each panel indicate the test voltage at which each channel was approximately 50% activated. B, amplitudes of the tail currents for WT (), A536W () and V535W () channels were normalized to the maximum current value (Imax). Data points shown represent averages (±S.E.M.) and the continuous lines represent the best fit of a Boltzmann function (see Methods). The values for G0, the work done at 0 mV, and zg, the effective number of gating charges moving across the membrane electric field, derived from the Boltzmann fits to these data are summarized in Table 1. C, bar graph showing G0 (i.e. G0=G0 (mutant) –G0 (WT)) for each mutant. Five mutants (K525W, A527W, R528W, V533W and R534W) caused minimal change in G0 (defined as G0 < 1 kcal mol–1, dashed line).
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It has been suggested that mutation of residues to tryptophan can introduce bias due to the large variations in the changes in side chain volume (Li-Smerin et al. 2000a), which does not appear to be the case here. The pattern of perturbation to the gating behaviour of HERG S4 tryptophan mutants was similar before and after correcting G0 values for changes in volume (see Table 1). The residues in which there was the largest change in |G0| when values have been corrected for change in size are the arginine residues. This is because there are relatively small changes in sidechain volume when mutating an arginine to a tryptophan (see eqn (7)).
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Effects of tryptophan mutants on rates of activation
Typical examples of currents recorded during protocols to measure rates of activation at 0 mV for WT, T526W and R531W channels are shown in Fig. 4A. T526W has a faster rate of activation at 0 mV whereas R531W has a slower rate of activation at 0 mV compared to WT channels. The majority of S4 tryptophan mutants had smaller time constants of activation (see Fig. 4B), and hence, faster rates of activation than WT channels. In particular, mutation of either of the lysine residues to tryptophan (K525W, K538W) caused marked acceleration of the rates of activation at 0 mV (see Fig. 4B).
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A, examples of rates of activation at 0 mV, measured using an envelope-of-tails protocol (see inset), for WT, T526W and R531W channels. The time constant of activation was obtained by fitting the envelope of peak tail currents with eqn (4) (see Methods). B, summary (mean ±S.E.M.) of time constants of activation for all S4 tryptophan mutants.
One concern with comparing the rates of activation at 0 mV, is that due to shifts in the voltage dependence of steady-state activation (see Fig. 3) the electrochemical potential for activation (chemical, G0, plus electrostatic, –zgEF) at any given voltage will be different for each mutant. We therefore measured the rates of activation for all the S4 tryptophan mutants and WT channels at multiple voltages in the range +40 to +160 mV and plotted the time constants of activation against the total electrochemical potential (–(G0–zgEF); see Fig. 5A. From these data we interpolated a value for the time constant of activation for each mutant channel at an electrochemical potential of 7 kcal mol–1 (dashed line, Fig. 5A and see Supplemental material Fig. S1 and Table S1) and then calculated a value for G of activation (see Fig. 5B; Table 2). We chose 7 kcal mol–1, as this was a value at which all channels would be fully activated and where we had data for all mutants. At an electrochemical potential of 7 kcal mol–1, the values of Gact showed a periodicity with the 1st, 3rd, 4th, 7th, 10th, 11th and 14th residues having either a positive or very small value for G. This pattern of perturbation to Gact was not altered when the values for Gact were corrected for changes in sidechain volume (see Table 2).
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A, time constants of activation for WT and S4 tryptophan mutant channels were obtained at voltages in the range 0 to +160 mV, and are plotted versus the total electrochemical potential (see Methods), i.e. –(G0–zgEF). Error bars are omitted for purposes of clarity. The mean data (±S.E.M.) for the interpolated time constants of activation calculated for a driving force at 7 kcal mol–1 for each mutant in at least n= 4 separate oocytes is shown in Supplemental material Table S1. B, plot of G. At an equivalent electrochemical potential of 7 kcal mol–1, there is a periodicity in the perturbation of G with the 1st, 3rd, 4th, 7th, 10th, 11th and 14th residues having either a positive or very small value for G.
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Effects of tryptophan mutants on rates of deactivation
Typical examples of tail currents recorded at –80 mV for WT, L539W and V535W channels are shown in Fig. 6A. Current traces have been normalized to the peak tail current to facilitate comparison of the rates of deactivation. In the traces shown in Fig. 6A, L539W channels deactivate more slowly than WT, whereas V535W channels deactivate more rapidly. The rates of deactivation at –80 mV for all S4 tryptophan mutants varied from 35 ± 2 ms for V535W to 1093 ± 6 ms for R528W compared to 198 ± 1 ms for WT HERG (see Fig. 6B). The problems with comparing rates of activation between mutant channels at a given voltage (see above) also applies to comparing rates of deactivation at a given voltage. We therefore measured rates of deactivation at multiple voltages in the range –60 to –190 mV and have plotted the rates of deactivation against the electrochemical potential for deactivation (see Fig. 7A). To quantify the effect of each mutant on the rate of deactivation at an electrochemical potential of 7 kcal mol–1, we calculated a perturbation energy Gdeact (eqn (5)) for each mutant relative to WT (see Fig. 7B; Table 2). There is a clear periodicity of perturbation to Gdeact at an electrochemical potential of 7 kcal mol–1 with the 1st, 2nd and then the 6th, 9th, 12th, 13th and 15th residues showing significantly more negative Gdeact values than the other residues (see Fig. 7D).
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A, typical examples of tail currents recorded at –80 mV for WT, L539W and V535W following a step to +40 mV to activate the channels (voltage protocol shown in inset). Traces have been normalized to the peak tail current to facilitate comparison of the rates of deactivation. In the traces shown, the currents for L539W decay more slowly than for WT, which in turn is slower than for V535W. B, summary (mean ±S.E.M.) of time constants of deactivation for all S4 tryptophan mutants at –80 mV.
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A, time constants of deactivation for WT and S4 tryptophan mutant channels were obtained at voltages in the range 0 to –190 mV, and are plotted versus the electrochemical potential, i.e. –(G0–zgEF). B, perturbation of deactivation, G, calculated using eqn (5) (see Methods), for time constants of deactivation calculated at an equivalent electrochemical potential of 7 kcal mol–1.
Are charged residues in the S4 of HERG exposed to a lipid or aqueous environment
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Many previous studies have suggested that the charged residues in S4 interact with an aqueous cavity (see, e.g. Gandhi et al. 2003; Laine et al. 2003; Ahern & Horn, 2004; Bell et al. 2004; Starace & Bezanilla, 2004; Tombola et al. 2005). Conversely, the crystal structures of KvAP and Kv1.2 suggested that some of the positively charged residues may interact with lipid (Jiang et al. 2003a; Long et al. 2005a). Our data, showing that tryptophan mutants of S4 charged residues are all tolerated, suggest that in HERG K+ channels some of the charged residues may be exposed to lipid in at least one conformational state of the channel. In previous studies we mutated each of the charged residues in the S4 domain of HERG (K525, R528, R531, R534, R537) to glutamine (Subbiah et al. 2004b) and we have extended this to K538 (see Fig. S2, Supplemental material). In all instances the glutamine substitution caused a similar or greater perturbation to G0 than a tryptophan substitution (see Fig. 8). The most significant differences between the tryptophan and glutamine substitutions occurred for R528 and R531 (see Supplemental material Table S1). Taken together, the data from the glutamine and tryptophan mutants suggest that the charged residues in the S4 domain (and in particular R528 and R531) are likely to interact with a lipid environment in one or more conformational states of the channel and so more readily permit substitution with the bulky hydrophobic tryptophan sidechain than glutamine.
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Comparison of perturbation to G0 for tryptophan (filled bars) and glutamine (open bars) mutants of the charged residues in the S4 domain of HERG. Glutamine substitution compared to tryptophan substitution of S4 charged residues caused a similar or greater perturbation to G0. The most significant differences observed between the tryptophan and glutamine substitutions were at R528 and R531.
Discussion
HERG K+ channels exhibit unusual kinetics characterized by slow activation and deactivation compared to rapid inactivation and recovery from inactivation. Previous studies have shown that slow activation is caused by slow movement of the S4 domain (Smith & Yellen, 2002; Piper et al. 2003). The mechanisms by which the S4 domain moves slowly, however, remain to be determined. In this study we have shown that every residue in the HERG S4 domain (from L524 to L539) can be mutated to tryptophan and still produce functional channels. In previous studies where tryptophan-scanning mutagenesis was employed in inward rectifier K+ channels or the pore of the Shaker K+ channel, between 15 and 55% of residues were intolerant of tryptophan substitution (Choe et al. 1995; Collins et al. 1997; Hackos et al. 2002; Sukharev et al. 2003). From these data, it was concluded that the non-functional tryptophan mutants identified residues involved in tight protein–protein interactions, whereas the remaining residues probably faced the lipid. In marked contrast to these studies, only 2 out of 67 residues in the S1, S2 and S3 domains of the Shaker K+ channel did not tolerate substitution to tryptophan (Monks et al. 1999; Hong & Miller, 2000). Based on these data Miller and colleagues concluded that the S1–S3 segments had a peripheral location, adjacent to lipid, and presumably surrounded and protected the S4 domain from the lipid. However, there will only be a periodicity in response to mutant scans if there are two environments that a domain is exposed to and the mutant residue perturbs interactions with one environment more than the other. Presumably, the two environments that S4 is exposed to are loose protein–protein interactions and a lipid environment. Therefore the observation that the S1–S3 segments can tolerate tryptophan substitutions at 65/67 locations indicates that these domains are adjacent to lipid and only loosely packed. By the same reasoning, we would have to conclude based on our experimental data for S4 tryptophan mutants of HERG that part the S4 domain in HERG is loosely packed within the voltage sensor and probably also faces the lipid. However, based on our data it is not possible to determine which residues of the HERG S4 domain face lipid. Furthermore, residues that interact with lipid may vary between different conformational states of the channel. Our observation that there was a helical periodicity to the effects of Tryptophan substitutions (see Figs 5B and 7B) is also consistent with previous studies indicating that the S4 domain must be -helical (Mulvey et al. 1989; Li-Smerin et al. 2000b; Jiang et al. 2003a; Long et al. 2005a).
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Our conclusion that the S4 domain must be loosely packed within the voltage sensor is somewhat at odds with the crystal structures of KvAP (Jiang et al. 2003a) and Kv1.2 (Long et al. 2005a), which indicate that the S4 and S3 domains are closely packed. However, given the relatively poor sequence homology between the S3b segment of HERG and KvAP (see Fig. 1C), one must be cautious with making structural predictions for HERG based on the KvAP and Kv1.2 voltage sensor structures.
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Slow activation of HERG
The slow activation of HERG is due to slow movement of the S4 domain (Smith & Yellen, 2002; Piper et al. 2003). This indicates that there must be a large energy barrier between the closed state and intermediate transition state. Mutants could therefore affect rates of activation either by stabilizing or destabilizing the closed or open states relative to each other or by destabilizing these states relative to an intermediate transition state.
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The residues that when mutated to tryptophan caused the largest acceleration in the rates of activation (after correcting for shifts in the voltage sensitivity of different mutants and alterations in sidechain volume; see column 3, Table 2) were K525, R528, R531, K538 and L539. In the case of K525, K538 and L539 these residues when mutated to tryptophan also showed marked acceleration of deactivation. Thus, one possibility is that mutation of K525, K538 and L539 results in stabilization of a transition state (and hence lower energy barrier) between the grouped closed and open states and so permit faster activation and faster deactivation. Further, K525W had minimal effect on the overall voltage dependence of the closed–open state equilibrium as measured by the Boltzmann distribution (see Fig. 4C). Thus in the case of K525W the effect of the mutation can be largely explained by stabilization of a transition state. K538W and L539W caused a modest destabilization of the closed state relative to the open state (negative G0, see Fig. 4C). Thus, in the case of K538 and L539, as well as stabilizing a transition state (see above) they must also be affecting the stability of the closed and/or open state.
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Mutation of R528 and R531 resulted in faster activation but no change or a slowing of deactivation (see Fig. 7). These residues correspond to R300 and R303 in Kv1.2 (see Fig. 1C) the residues that were shown to form salt bridges in the presumed open conformation of the Kv1.2 channel (Long et al. 2005a). If similar salt bridges exist in the open state of HERG then the R528 and R531 mutants would be predicted to destabilize the open state. Consistent with this R531W resulted in a significant destabilization of the open state relative to the closed state, as reflected in the Boltzmann distribution analysis of isochronal activation curves (see Fig. 4, Table 1). However, R528W did not. If the salt bridges were present in the transition state then one would expect the tryptophan mutants to destabilize the transition state and so slow activation (not observed) and deactivation (observed with R528W but not R531W). If the salt bridges are present in the closed state then one might expect tryptophan mutants to increase the rate of activation (observed with both R528W and R531W). Thus our data would be consistent with a salt bridge involving R528 present in at least one closed state and a transition state and a salt bridge involving R531 present in at least one closed state and one open state. It should also be noted that mutation of R531 or R528 to glutamine caused a greater perturbation to the overall open–closed state equilibrium than did mutation to tryptophan (see Fig. 7). These data suggest that in one or more conformational states R531 and R528 are likely to be exposed to lipid (although it is not necessarily the same conformational state for both residues). Overall, our data in conjunction with the data in the literature, are consistent with a model in which R528 and R531 participate in salt bridges in one or more but not all conformational states of the channel and are exposed to lipid in other conformational states of the channel.
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Scanning mutagenesis studies of the HERG S4 domain with glutamine identified K525Q, R528Q, R534Q (Subbiah et al. 2004b) and K538Q (see Supplemental material) as having significantly faster rates of activation than WT channels. In previous cysteine scanning (Zhang et al. 2004) and alanine scanning (Piper et al. 2005) mutagenesis studies of the HERG S4 domain, the rates of activation were not measured, so it is not possible to make direct comparisons with this study. However, in both the cysteine scanning and alanine scanning studies, mutations of K538 significantly destabilized the closed state of the channel, which would be consistent with faster rates of activation. Mutation of K525 also destabilized the closed state when mutated to cysteine (Subbiah et al. 2004b; Zhang et al. 2004) or was non-functional when mutated to alanine (Piper et al. 2005). The results for residues in the middle of S4 are more variable between the four studies. This suggests that the specific chemical properties of residues in the middle of S4 influence the activity of the channel. This in turn could be most easily explained by these residues contributing to specific protein–protein interactions within the voltage sensor domain, such as the putative salt bridges discussed above for R528 and R531.
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Together, the results from the four scanning mutagenesis studies suggest that the lysine residues at each end of the HERG S4 domain (K525 and K538) are important for stabilizing the closed state and thereby contributing to the slow activation of the channels. It is likely that residues in the middle of S4 may also be important for slow activation, with residues R528 and R531 key candidates for binding to residues in other transmembrane domains.
Slow deactivation of HERG
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The mutations that most perturbed the rates of deactivation (after correction for changes in electrochemical driving force and sidechain volume) were similar to those perturbing the rates of activation (see Supplemental material Table S1). In particular, mutating K525, R528 and K538 significantly affected rates of both activation and deactivation (as discussed above). There are however, some differences, e.g. mutating L524 and L532 had significant effects on deactivation but not activation and mutation of R531 had significant effects on activation but not deactivation. This is probably not surprising given that activation and deactivation are multistep processes and the measured rates of activation and deactivation are not necessarily measurements of the forward and reverse rates of the same step in the process. Thus, if mutation of L524 and L532 to tryptophan stabilizes the transition state for the rate-limiting step in the deactivation process but this same step was not rate limiting for activation, then the mutants would lead to faster deactivation but have minimal effect on activation.
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Models of voltage sensing
The conundrum of how charges can move across the transmembrane electric field, yet be shielded from the thermodynamic cost of being exposed to a hydrophobic lipid environment has puzzled biophysicists for many decades (Bezanilla, 2000). The sliding-helix model, in which it was proposed that the positively charged residues in S4 were cocooned in a proteinaceous jacket composed of the S1–S3 domains to protect them from the hydrophobic membrane interior, provided an elegant explanation for this conundrum (see, e.g. Keynes & Elinder (1999) for review). With minor modifications, the sliding-helix model has remained the accepted paradigm for the mechanism of voltage sensing, until very recently. However, data from the crystal structure of KvAP (Jiang et al. 2003a), supported by evidence from biotin–avidin accessibility studies (Jiang et al. 2003b) and electron microscopy studies (Jiang et al. 2004) of the KvAP channel, are incompatible with the sliding-helix model. MacKinnon and colleagues postulated an alternative model, the paddle model, in which the S4 domain lies at the periphery of the channel and is able to move freely across the lipid membrane in response to changes in membrane voltage (Jiang et al. 2003b). However, all the evidence, to date, in support of the hydrophobic cation model of voltage sensing has come from studies of the bacterial KvAP channel. Could it also explain voltage sensing in mammalian voltage-gated ion channelsThis is of particular relevance to cardiac biology as voltage-gated ion channels are central to the regulation of the heart beat. Furthermore, mutations in the voltage sensor domains of voltage-gated ion channels can predispose to an increased risk of cardiac arrhythmias (Clancy & Kass, 2005).
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Our data showing that every residue in the S4 domain of HERG can tolerate a tryptophan substitution places significant constraints on the orientation of S4 with respect to the remainder of the channel and the lipid bilayer. Our data suggest that the S4 domain must be loosely packed with respect to the rest of the channel and at least partially lipid exposed. The tolerance of tryptophan substitutions is consistent with a peripheral location for S4 as suggested in the paddle model proposed by MacKinnon and colleagues (Jiang et al. 2003b, 2004; Long et al. 2005b). However, it is not consistent with the traditional sliding-helix model where S4 is cocooned within a proteinaceous jacket composed of the S1–S3 domains (Keynes & Elinder, 1999). The tolerance of tryptophan substitutions, however, does not support a tight interaction between S4 and S3 as suggested in the paddle model (Jiang et al. 2003b; Long et al. 2005a).
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Recently, Starace & Bezanilla (2004) and Tombola et al. (2005) have suggested a modification to the sliding-helix model, known as the transporter model, in which the S1–S3 domains are loosely packed around S4 and changes in the transmembrane electric field, consequent to movement of S1–S3 relative to S4, result in the effective movement of S4 charges across the membrane. The tolerance of tryptophan substitutions at all residues in the S4 domain of HERG is consistent with loose packing of the S4 domain as proposed in this transporter model.
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