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1 Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298, USA
2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
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Abstract |
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(Received 27 June 2005;
accepted after revision 2 September 2005;
first published online 8 September 2005)
Corresponding author G.-N. Tseng: Department of Physiology, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA 23298, USA. Email: gtseng{at}hsc.vcu.edu
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Introduction |
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The hERG channel shares structural design with other Kv channels. Each functional channel has four subunits, and each subunit is composed of a voltage-sensing domain (S1–S4) and a pore domain (S5–pore loop–S6) (Fig. 1A). hERG differs from many K+ channels in having a longer S5–P linker (43 aa versus 12–23 aa, Fig. 1B). We have performed a cysteine (Cys) scanning mutagenesis analysis of hERG's S5–P linker (Liu et al. 2002). Cysteines introduced into 14 positions in the centre of the hERG S5–P linker, from 583 to 597 (excluding 595; cysteine substitution at this position produces a non-functional channel) can form disulphide bonds. Disulphide bond formation produces a uniform mutant phenotype: disruption of the fast-inactivation process and the pore's K+ selectivity (Liu et al. 2002). Thus, the central portion of the S5–P linker in the hERG channel can engage in interactions with other channel domains, or with its counterparts, to allow disulphide bond formation. Furthermore, its peptide backbone conformation or interaction with other channel domains or counterparts is important for the fast-inactivation process as well as the pore's K+ selectivity.
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Why is the central portion of the S5–P linker so critical for the fast-inactivation process and K+ selectivity of the hERG channel? Inactivation in the hERG channel is similar to the C-type inactivation in the Shaker channel (Hoshi et al. 1991; Smith et al. 1996; Herzberg et al. 1998): inactivation results from conformational changes around the selectivity filter that prevent ion conduction through the pore. The channel pore's selectivity for K+ ions is also mainly determined by the selectivity filter (Fig. 1B). In the available K+ channel crystal structures (Doyle et al. 1998; Jiang et al. 2002; Jiang et al. 2003), the turrets (corresponding to hERG's S5–P linker) are immersed in the extracellular solution, making no direct contact with the selectivity filter (Fig. 1C). However, our data described above suggest that this may not be the case for hERG. In this study, we take several approaches to examine the secondary structure of the 583–597 segment and to explore its interaction with other channel domains: (1) circular dichroism (CD) spectral analysis and spatial structure calculations based on nuclear magnetic resonance (NMR) data are used to directly probe the secondary structure and side chain mobility of a synthetic hERG S5–P linker peptide that includes the 583–597 segment and its flanking regions, and (2) site-specific mutations in conjunction with biochemical and electrophysiological analysis are used to probe side chain interactions in this segment of an intact channel and how these interactions impact on channel function.
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Methods |
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Peptide synthesis. A 25-mer ‘hERG S5–P linker’ peptide corresponding to hERG residues 578–603 was synthesized by Obrigen (San Diego, CA, USA). The peptide was purified by reverse phase HPLC and composition of the purification product was verified by mass spectroscopy.
CD spectroscopy. Sample of hERG S5–P linker peptide (74 µM) in 4.44 mM diphenylamine carboxylic acid (DPC) (peptide/DPC = 1/60) or in 5.92 mM sodium dodecyl sulphate (SDS) (peptide/SDS = 1/80) was prepared in H2O and pH was titrated to 3.5. CD spectra were obtained using spectropolarimeter J-715 (Jasco, Easton, MD, USA) in the wavelength range 185–250 nm at 22°C with a 0.02 cm quartz cell.
NMR spectroscopy.
NMR sample of 0.6 mM hERG S5–P linker peptide in 10% D2O or of 0.35 mM peptide in 21 mM DPC (peptide/DPC = 1/60, in 10% D2O) was used. 1H NMR spectra (double quantum-filtered correlated spectroscopy (DQF-COSY) (Rance et al. 1983), total correlation spectroscopy (TOCSY) (Bax & Davis, 1985) with mixing times (
m) of 40 and 80 ms, and Overhauser enhancement spectroscopy (NOESY) (Jeener et al. 1979) with m of 100 and 200 ms) were acquired using a 600 MHz spectrometer (Unity 600, Varian, Palo Alto, CA, USA) at 30°C, pH 3.5. NMR data were processed with VNMR (Varian software), and analysed with the XEASY program (Bartels et al. 1995). Proton resonance assignment of hERG S5–P linker peptide in DPC was performed by standard procedure (Wuthrich, 1986) using the XEASY program. Three hundred and eighty-six cross-peaks in the NOESY (m= 100 ms) spectrum of peptide in DPC were assigned unambiguously. Cross-peak intensities of peptide in DPC were measured in 100 ms NOESY spectrum using the XEASY program. The interproton distance constraints were derived from NOESY cross-peak volumes via ‘1/r6’-calibration, using the CALIBA module of the DYANA program (Guntert et al. 1997). Vicinal spin–spin coupling constants 3JHN-C
H were determined from the fine structure along the 
2 axis of the non-overlapped NOESY cross-peaks between the corresponding amide proton (at the 2 frequency) and any proton of other residues (at the 1 frequency). 3JHC-CßH values were measured in DQF-COSY or TOCSY (m= 40 ms) spectra. Torsion angle constraints were derived from spin–spin coupling constants and local nuclear Overhauser enhancement (NOE) distance constraints using GRIDSEARCH module of the DYANA program (Guntert et al. 1997).
Structure calculation. Spatial structure calculation of hERG S5–P linker peptide in DPC was performed using the DYANA program (Guntert et al. 1997). Twenty best structures were selected based on the criterion of small DYANA target function out of 100 calculated structures conforming to experimental constraints. Hydrogen bonds were identified from an analysis of these structures using distance and angle criteria (Baker & Hubburd, 1984). Visual analysis of structures and preparation of figures were performed using the MOLMOL program (Koradi et al. 1996).
Mutagenesis and oocyte voltage clamp experiments
The cysteine mutations of the hERG channel have been described before (Liu et al. 2002). Oocytes were isolated from Xenopus laevis. Animal usage is reviewed annually, and approved, by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Isolated oocytes were incubated in an ND96-based medium (mM: NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, Hepes 5, sodium pyruvate 2.5, pH 7.5, supplemented with 10% horse serum and penicillin/streptomycin) at 16°C. Five to twelve hours after isolation, each oocyte was injected with cRNA solution using a Drummond digital microdispenser. Oocytes were incubated in the above medium at 16°C, and studied 2–4 days after cRNA injection. Membrane currents were recorded from whole oocytes using the ‘2-cushion pipette’ voltage clamp method in a low-Cl ND96 solution (Cl– in ND96 replaced by methanesulphonate) to reduce interference from endo-genous Cl– channels (Schreibmayer et al. 1994). Voltage clamp was done at room temperature (24–26°C) with OC-725B or OC-725C amplifier (Warner Instruments, MA, USA). Voltage clamp protocol generation and data acquisition were controlled by pCLAMP5.5 via a 12-bit D/A and A/D converter (DMA, Axon Instruments, Union City, CA, USA). Current data were low-pass filtered at 1 kHz (Frequency Devices Inc., Haverhill, MA, USA) and stored on disks for off-line analysis. CdCl2 stock solution (1, 10 or 100 mM) was added to the bath solution to reach desired final concentrations (0.2–100 µM).
Immunoblot and immunocytochemistry
Oocytes were injected with cRNA and incubated as described above. Three days after cRNA injection, oocytes (15–25 oocytes per group) were incubated with 20 mMN-ethylmaleimide (NEM) in Tris–EDTA (TE) buffer containing protease inhibitor cocktail (Sigma) at room temperature for 15 min. Oocytes were then homogenized with a loose-fit glass grinder on ice. The homogenates were centrifuged at 3000 g for 15 min to remove debris, and the supernatants were overlaid on 15% sucrose cushion (in TE buffer containing 20 mM NEM and protease inhibitor cocktail) and centrifuged at 175 000 g for 75 min at 4°C. The pellets (enriched membrane fractions) were resuspended in phosphate-buffered saline (PBS) containing 103 mM KCl and protease inhibitor cocktail and solubilized by incubating with 1% SDS at room temperature for 2 h followed by sonication. Each sample was divided into two aliquots. One was added 2x sample buffer containing 125 mM DTT (reducing) and the other was added 2x sample buffer without DTT (non-reducing) Both were boiled for 5 min before loading on non-reducing SDS–5% polyacrylamide gel. After electrophoresis, proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Amersham, Piscataway, NJ, USA). The PVDF membrane was blocked in PBS with 5% non-fat dried milk–0.1% Tween 20 for 1 h at room temperature and then incubated with anti-erg1 antibody (Alomone Labs Ltd, Jerusalem, Israel) at 4°C overnight. This was followed by three 10 min rinses in PBS with 0.1% Tween 20. The membrane was then incubated with alkaline phosphatase-conjugated secondary antibody (Amersham) for 1 h at room temperature. Immunoreactivity was visualized using an enhanced-chemiluminescence (ECL) detection kit (Amersham).
Oocytes injected with cRNAs or uninjected control oocytes were frozen in Optimal Cutting Temperature compound (OCT). Thin (10-µm) sections were cut with a cryostat, fixed in 4% formaldehyde, permeabilized with 2% Triton X-100, blocked by 4% goat serum, incubated with anti-erg1 antibody (Alomone), rinsed, incubated with an Alexa 568-conjugated anti-rabbit antibody (Molecular Probes, Eugene, OR, USA), mounted with ProLong antifade agent (Molecular Probes), and viewed using a Zeiss 510 confocal microscope.
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Results |
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A synthetic peptide corresponding to amino acids 578–603 of hERG was used in these experiments. CD spectra in Fig. 2A indicate that this hERG S5–P linker peptide was unstructured in a pure aqueous solution, but possessed 
30% helical structure in detergent micelles (DPC or SDS). Figure 2B summarizes NMR data used to locate the peptide's helical structure when placed in DPC micelles. Spatial structure calculations showed that the N-terminal (corresponding to hERG residues 578–582) and the C-terminal (594–603) regions were unstructured. However, the segment from 583 to 593 was helical, with 583–590 forming an -helix and 591–593 forming a 310 helix (Fig. 2C and D). Although we cannot rule out the possibility of solvent-induced formation of an -helical structure, the NMR analysis in conjunction with our previous functional data (Liu et al. 2002) makes a compelling case for the notion that in a functional hERG channel the 583–593 segment can form an -helix at least in some gating states of the channel.
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1 values for W585 and L589, as well as D591, indicates that these side chains may have increased mobility (Fig. 2C). Western blot analysis reveals that cysteine side chains introduced into the 583–597 segment can form intersubunit disulphide bonds
Cysteine side chains introduced into all positions in the 583–597 segment (excluding non-functional K595C) can spontaneously form disulphide bonds (Liu et al. 2002). Where are their ‘partner’ cysteine side chains? To answer this question, we first needed to determine whether these introduced cysteine side chains form inter- or intra-subunit disulphide bonds. Figure 3A depicts possible scenarios. An introduced cysteine may form a disulphide bond with its counterpart or a native cysteine from a neighbouring subunit (intersubunit disulphide, forming a dimer of 254 kDa), or with a native cysteine from the same subunit (intrasubunit disulphide, remaining as monomer of 127 kDa). To determine the nature of disulphide bonds formed by the cysteine side chains introduced into the 583–597 segment, we applied Western blot analysis to channel proteins expressed in oocytes. To avoid non-specific disulphide bond formation, live oocytes were treated with 20 mM NEM to protect free thiol groups before homogenization (see Methods). Oocyte homogenization and membrane fraction isolation were carried out under non-reducing conditions. The final product was divided into two aliquots, one treated with DTT and the other maintained in non-reducing condition, to test whether DTT can alter the banding pattern of channel proteins on the Western blot.
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The above controls confirm the validity of using Western blot analysis on channel proteins expressed in oocytes to detect intersubunit disulphide bond formation. For the cysteine-substituted mutants in the 583–597 segment, G590C did not produce sufficient protein for Western blots (3 attempts). Therefore, this mutant, along with the non-functional K595C, was not included in the Western blot experiments. Figure 3B shows that under non-reducing conditions all 13 cysteine-substituted channel proteins migrated as two bands: one dimer band and one monomer band (– DTT, left of each pair of channel lanes). All the dimer bands disappeared after DTT treatment (+ DTT, right of each pair of channel lanes). Therefore, cysteine side chains introduced into positions 583–597 (excluding 590 and 595) were all capable of forming intersubunit disulphide bonds.
We used the ratios of dimmer : monomer band intensities as an indicator of how readily the introduced cysteine side chains can form intersubunit disulphide bonds, or how stable such intersubunit disulphide bonds are. The data are summarized in Fig. 3C (numbers in parentheses denote numbers of experiments using different batches of oocytes). Overall, there was a trend of decreasing ratio of dimmer : monomer band intensities from the N-terminus to the C-terminus of the 583–597 segment (denoted by the dashed curve), although N588C, L589C and Q592C formed stronger dimer bands than suggested by the trend.
Cysteines introduced into four positions along the 583–597 segment can form intersubunit disulphide bonds with their counterparts from neighbouring subunits
We further explored whether the intersubunit disulphide bonds were formed between counterparts of introduced cysteine side chains, or between introduced and native cysteine side chains. Four positions with a high tendency of dimer formation were chosen for this analysis: 584, 585, 588 and 589. There are a total of 24 native cysteine side chains in each hERG subunit. Among them, five are in the extracellular region or in the transmembrane segments, and thus could potentially form disulphide bonds with the introduced cysteines. These are C445 and C449 in the S1–S2 linker, C555 and C566 in S5, and C643 in S6. All five native cysteines were substituted with alanine, creating a ‘Cys-removed’ hERG background (labelled as ‘– Cys background’ in Figs 4 and 5). Cysteine side chains were then reintroduced into this Cys-removed background at 584, 585, 588 and 589.
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Figure 5B depicts Western blot images of G584C, W585C, N588C and L589C proteins in the Cys-removed background run on a non-reducing gel. The same mutants in the WT background were included as positive control for dimer formation, while the background channel proteins without any introduced cysteines were included as negative control (monomer bands only). All four cysteine-substituted mutants in the Cys-removed background showed clear dimer bands as well as monomer bands, similar to the banding pattern of corresponding cysteine-substituted mutants in the WT background. Similar observations were obtained in multiple experiments (numbers shown in Fig. 5D, from different batches of oocytes). Furthermore, as expected DTT treatment abolished the dimer bands (Fig. 5C). Therefore, cysteine side chains introduced into positions 584, 585, 588 and 589 can form disulphide bonds with their counterparts from neighbouring subunits.
Cysteine side chains at 584, 585, 588 and 589 can coordinate high-affinity Cd2+ binding
Disulphide bond formation can trap two cysteine side chains in a bonded state even when the two are apart by as far as 15 Å in flexible proteins (Careaga & Falke, 1992). On the other hand, the formation of a Cd2+ bridge has a more stringent requirement for spatial distance between cysteine side chains: distance between Cß atoms should be < 5 Å (Castagnetto et al. 2002). Furthermore, the Cd2+ affinity depends on the number of thiol side chains that coordinate the Cd2+ bridge (Rulisek & Vondrasek, 1998). A tight Cd2+ bridge formed between two or more closely positioned cysteine side chains cannot be broken by removing ambient Cd2+ ions. A divalent cation chelator, e.g. EDTA, is needed to remove the bound Cd2+ ion. Therefore, to ascertain that cysteine side chains at positions 584, 585, 588 and 589 were indeed close to their counterparts from neighbouring subunits, we tested the effects of Cd2+ and the reversibility of these effects upon washout of Cd2+ ions. To obtain direct functional readouts under voltage clamp conditions, experiments on G584C, W585C and L589C were conducted using the WT background. For N588C, both WT background and Cys-removed background were studied since both produce sizable currents. This gave us an opportunity to evaluate whether native cysteines play any role in coordinating Cd2+ binding to this cysteine-substituted mutant.
Since Cd2+ can affect the WT hERG channel (Ho et al. 1999; Johnson et al. 1999), we first examined the concentration dependence and reversibility of Cd2+ effects on WT hERG. WT hERG was not sensitive to 1 µM[Cd2+]o (n= 6, one shown in Fig. 6A). At 10 µM, Cd2+ accelerated deactivation, shifted V0.5 of activation by 15.6 ± 1.2 mV, and modestly reduced the peak amplitude of tail currents (by 14 ± 3%, n= 5). At 100 µM, Cd2+ suppressed the tail current amplitude by 70%, further accelerated deactivation, and caused a prominent positive shift of V0.5 by 34 mV (n= 2). Even with 100 µM, these Cd2+ effects on WT hERG were readily reversible upon washing out of Cd2+ (Fig. 6A). Therefore, native cysteines in the WT hERG channel were too far apart to coordinate a tight Cd2+ binding site on their own.
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Cysteine side chains introduced into another position on the hydrophilic face of the S5–P helix, 584, could also coordinate a high-affinity Cd2+ binding site (Fig. 7). The effects could not be reversed by washing out Cd2+, but required EDTA to chelate the bound Cd2+ ions. The potency of Cd2+ to suppress G584C appeared independent of [K+]o (IC50= 0.65 ± 0.08 µM and 1.69 ± 0.81 µM, in 98 and 2 mM[K+]o, respectively, n= 4 each, P= 0.343).
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Discussion |
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NMR structures of the isolated S5–P linker peptide: implications for the S5–P linker structure in a functional hERG channel in the cell membrane
Our NMR analysis indicates that the hERG S5–P linker peptide (residues 578–603) can adopt a helical structure in its central portion, with residues 583–590 forming an -helix and residues 591–593 forming a 310 helix. This prediction is similar, although not identical, to a previous NMR analysis of a longer hERG S5–P peptide (residues 570–611) (Torres et al. 2003). The CD data suggest that if the hERG S5–P linker is completely immersed in the extracellular aqueous phase, it is likely to be unstructured. When it is in detergent micelles that mimic the cell membrane environment, it can adopt a helical structure. Our previous functional data suggested that the 583–594 segment has a helical structure (Liu et al. 2002). However, in a functional channel the hERG S5–P linker is not likely to be embedded in the membrane lipid bilayer. This casts doubt as to whether the S5–P linker can adopt an -helical structure in a functional channel. The putative S5–P helix has a hydrophobic face occupied by high-impact positions (W585, L586, L589 and I593) and a hydrophilic face occupied predominantly by intermediate-impact positions (H587, N588, D591 and Q592) (Fig. 8A). It is possible that in a functional channel the central portion of the S5–P linker forms an amphipathic helix lying in the interface between hydrophilic extracellular aqueous solution and hydrophobic channel protein surface, with the hydrophobic residues on the S5–P helix interacting with channel surface domains to stabilize the -helical structure. Such intimate interactions between channel domains are consistent with the ‘high-impact position’ status of these hydrophobic residues on the S5–P helix (Fig. 8A). In this scenario, the hydrophilic residues face the extracellular aqueous solution, where cysteine substitution has little effects on channel function. However, MTSET modification of these cysteine side chain can reduce current amplitude, or in the case of G584C, disrupt the fast-inactivation process and the pore's K+ selectivity. Since cysteine side chains at the intermediate-impact positions can also form disulphide bonds, the hydrophilic face of the S5–P helix must be able to transiently interact with other channel domains or its counterparts in some gating conformations. Taken together, our data suggest that the S5–P linker is not a static structure. Instead, the S5–P helix may rotate around its long axis to allow disulphide bond formation along both its hydrophobic and hydrophilic faces. These helices may also swing along the channel surface to allow cysteine side chains at positions 584, 585, 588 and 589 to come close to their counterparts to form disulphide bonds or coordinate high-affinity Cd2+ binding (distance between Cß atoms < 5 Å).
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Our previous data suggest that the N-terminus of the S5–P helix is close to the pore entrance and to counterparts from other subunits (Liu et al. 2002). We also propose that the S5–P helix is orientated with its C-terminus pointing away from the pore entrance. This arrangement is consistent with the trend of intersubunit disulphide bond stability suggested by Fig. 3C: the N-terminal end of S5–P helix is closer to each other than the C-terminal end and can more readily form intersubunit disulphide bonds. If the S5–P helices are arranged around the central pore in 4-fold symmetry, then the distance between cysteine side chains at equivalent positions on the S5–P helices is shorter between adjacent subunits than between diagonal subunits. Although we do not have high-resolution data to definitely conclude one way or the other, the available information does suggest that these cysteine side chains are more likely to from intersubunit disulphide bonds with counterparts from adjacent subunits than from the diagonal subunit. Data from G584C also support this scenario. This channel manifests WT-like behaviour when the thiol side chains are in reduced state, but switches to the mutant behaviour (disruption of fast inactivation and K+ selectivity) when the thiol side chains form disulphide bonds. Therefore, we can track disulphide bond formation during the course of an experiment. Figure 7A shows that after DTT washout, G584C gradually switches to the mutant behaviour, indicating disulphide bond formation. This process of ‘phenotype-switch’ reaches a steady state in about 30 min. Adding Cd2+ 1 µM at this point can effectively suppress the current, and washing out Cd2+ does not reverse the suppressing effect until EDTA is applied. This indicates that Cd2+ is coordinated by at least two cysteine side chains in close proximity (although histidine side chains can also coordinate Cd2+ ions). Although this observation represents the averaged behaviour of all G584C channels in the oocyte cell membrane, the data are consistent with the notion that disulphide bonds are formed between 584C from two adjacent subunits (thus allowing current through the pore). Furthermore, formation of one disulphide bond within a channel between two adjacent subunits does not perturb the outer mouth conformation significantly, so that the remaining two free thiol groups can still coordinate a high affinity Cd2+ binding site.
Why is the S5–P linker so critical for the fast-inactivation process of the hERG channel?
In Fig. 8B, we propose three models for how S5–P helices in a functional hERG channel interact with each other, and how these S5–P helices may contribute to the channel's fast-inactivation process. Although a transition of the S5–P linker conformation between helical and coil is possible during hERG gating, this possibility is not included in the simplified models depicted in Fig. 8B. In the left panel of Fig. 8B, the S5–P helices are orientated perpendicular to the plane of the cell membrane. Relatively small asymmetric rotations of S5–P helices along their long axes can allow intersubunit disulphide bond formation between cysteine side chains at equivalent positions from adjacent or even diagonal subunits. These S5–P helices come close to each other during channel inactivation, preventing K+ ion flux through the pore by steric hindrance or by a hydrophobic seal formed between hydrophobic residues along the S5–P helices. In the middle panel, the S5–P helices are orientated parallel to the plane of the cell membrane. Asymmetric rotations of S5–P helices along their long axes, as well as relatively large-scale sideway swinging motion along the channel surface, are needed to allow cysteine side chains at equivalent positions of adjacent subunits to form intersubunit disulphide bonds. Channel inactivation occurs when all four helices point their N-terminal positive helical dipole (Hol, 1985) toward the pore entrance, creating a local electrostatic potential unfavourable to K+ ion flux through the pore. In the right panel, the S5–P helices are tilted and sink into the channel pore to interact with the selectivity filter. Asymmetric rotations along their long axes as well as small-scale sideways swinging are needed to allow intersubunit disulphide bond formation between cysteine side chains at equivalent positions of adjacent subunits. Channel inactivation is envisioned to be due to two factors: (a) local electrostatic potential due to the N-terminal positive helical dipole of the four S5–P helices (Hol, 1985), and (b) interactions between the S5–P helices and the pore-loops, causing conformational changes around the selectivity filter that shut down K+ flux through the pore.
The advantage of the first model is that relatively small molecular motions can allow intersubunit disulphide bond formation between counterparts from adjacent or diagonal subunits. However, the disadvantage of this model is that it does not explain how the S5–P helices are stabilized in an aqueous environment. In the second and third models, the S5–P helices are stabilized by the interaction between residues on its hydrophobic face and the channel surface domain. The third model has the added advantages over the second model in that smaller sideways swinging motion allows intersubunit disulphide bond formation between cysteines at equivalent positions of two adjacent subunits, and the proposed interaction between the S5–P helix and the pore loop can better explain why the pore's K+ selectivity is disrupted by mutations at high-impact positions along the S5–P helix. We have proposed that relative to the Shaker channel, there are fewer hydrogen bonds formed between residues around the outer mouth of the hERG channel (Fan et al. 1999). This may result in a ‘floppy’ outer mouth structure, able to accommodate the insertion of the S5–P helices as proposed in the third model.
Technical consideration: validity of using cysteine-substituted mutants or cysteine-removed background channels to probe the structure–function relationship of hERG's S5–P linker
A critical issue for data interpretation in these experiments is whether the cysteine-substituted mutants maintain the native conformation of the channel's outer vestibule. This is the case for cysteine substitution at intermediate-impact positions, because these channels maintain the WT phenotype as long as the thiol side chains are in the reduced state. For cysteine substitution at the high-impact positions, although the inactivation process and K+ selectivity are disrupted even when the thiol side chains are reduced, 5 of the channels (L586C, N588C, L589C, D591C and I593C) maintain a high sensitivity to a peptide toxin, ErgTx1, similar to that of the WT hERG channel (mean IC50 range 2.3–10.7 nM, versus 7.2 nM for WT hERG) (Pardo-Lopez et al. 2002). Since such a high toxin binding affinity requires the maintenance of multiple contact points between toxin and the channel's outer vestibule, the data argue that these five cysteine-substituted mutants at the high impact positions do not drastically alter the outer mouth conformation. The remaining two cysteine substituted mutants (W585C and G590C) manifest markedly reduced ErgTx1 binding affinity (IC50 > 100 nM) (Pardo-Lopez et al. 2002). They are therefore less informative because of the uncertainty as to whether they maintain the native conformation of the outer vestibule.
Currents through channels in the Cys-removed background (5 native cysteines replaced by alanines, Fig. 4 legend) are very small or not detectable. This is likely to be due to the removal of C566 in S5, because removing the other four native cysteines, singly or in combination, does not significantly reduce the current amplitude (data not shown). Although the mechanism for the detrimental effect of C566A on hERG channel function is not clear, we argue that the transmembrane topology of channels in the Cys-removed background is maintained as the WT hERG. This is based on the observation that channels in the Cys-removed background can traffic to the oocyte cell surface, similar to channels in the WT background (Fig. 4B). This is possible only if these channel proteins are correctly folded so that they can exit the ER checkpoint. It is important to point out that in the oocyte Western blot experiments, all free thiol groups in intact and functional channels are blocked by 20 mM NEM before homogenization (see Methods). Disulphide bonds detected in Western blot experiments represent those formed in the in situ intact channels. Therefore, our data rule out the possibility that for channels in the Cys-removed background the remaining native cysteine side chains (in the cytoplasmic domain) can form disulphide bonds with those cysteine side chains introduced into the extracellular S5–P linker and interfere with data interpretation.
Our data cannot distinguish between two possibilities: cysteine side chains introduced into the 583–597 segment form disulphide bonds with specific partners, or with multiple partners in different gating conformations. In the Shaker channel, cysteine side chains introduced into several positions in S4 can form disulphide bonds with cysteine introduced into the same position of S5 in a gating state-dependent manner (Gandhi et al. 2003). Furthermore, whether cysteines introduced into positions other than 584, 585, 588 and 589 can form intersubunit disulphide bonds with counterparts from neighbouring subunits needs to be tested.
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denote overlapped cross-peaks. 3JN
and 
) and side chain torsion angles (
