Address correspondence to Gea-Ny Tseng, Department of Physiology, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA 23298. Fax: (804) 828-7382; E-mail: gtseng{at}hsc.vcu.edu
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ABSTRACT |
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Key Words: rapid delayed rectifier K channel LQT2 cysteine-scanning mutagenesis oocyte expression
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INTRODUCTION |
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The fast inactivation process of HERG/IKr shares important features with those of "C-type inactivation" in the Shaker channel (Hoshi et al., 1991; Yellen et al., 1994
; Smith et al., 1996
). These include the sensitivity to mutations introduced to the outer mouth region of the channel, slowing of the inactivation process by an outer mouth blocker (e.g., TEA), or by an increase in pore occupancy by permeant ions (e.g., K+) (Hoshi et al., 1991
; Yellen et al., 1994
; Smith et al., 1996
). Shaker's C-type inactivation has been well studied. It is due to conformational changes around the outer mouth, that constrict the pore or change the pore's selectivity in such a way that K+ flux is prevented (Yellen et al., 1994
; Liu et al., 1996
; Starkus et al., 1997
). The C-type inactivation process in the Shaker channel and in other voltage-gated K (Kv) channels for which C-type inactivation has been described (Kv1.3 [Panyi et al., 1995
], Kv1.4 [Rasmusson et al., 1995
], Kv1.5 [Fedida et al., 1999
], and Kv2.1 [Klemic et al., 1998
]) share two common features: slow kinetics and a lack of intrinsic voltage dependence. The slow kinetics is at least partially attributed to hydrogen bonds formed around the outer mouth of the pore (Doyle et al., 1998
; Larsson and Elinder, 2000
; Ortega-Saenz et al., 2000
). These hydrogen bonds stabilize the outer mouth in the open state, resisting collapse during C-type inactivation (Larsson and Elinder, 2000
; Ortega-Saenz et al., 2000
). The trigger for C-type inactivation in Shaker and related Kv channels is believed to be the S4 movements induced by membrane depolarization (Gandhi et al., 2000
). There-fore, C-type inactivation in these channels is not intrinsically voltage sensitive, but derives its voltage sensitivity from a coupling to the voltage-dependent activation process. C-type inactivation in the HERG channel is extremely fast, and is intrinsically voltage-sensitive: it can occur at a time scale and in a voltage range with little or no channel activation (Sanguinetti et al., 1995
; Rasmusson et al., 1998
). What is the structural basis for these unique features of C-type inactivation in the HERG channel?
Fig. 1
shows an alignment of amino acid sequences that line the outer vestibule (S5-P and P-S6 linkers) and the outer portion of the pore (pore or P-loop) of HERG, KcsA (the bacterial proton-gated K channel whose crystal structure has been solved [Doyle et al., 1998]), and major classes of Kv channel. This comparison reveals two unique features of the HERG sequence. First, amino acids that are implicated in forming hydrogen bonds that stabilize the Shaker outer mouth in the open state (E418, W434, and Y445, highlighted by gray shade in Fig. 1) are well conserved among Kv channels as well as in KcsA, but are missing in HERG. This reduced hydrogen bonding capability in HERG may make its outer mouth more flexible, easier to collapse by conformational changes triggered by membrane depolarization. This can contribute to the uniquely fast C-type inactivation kinetics in the HERG channel. Second, the S5-P linker in the HERG channel is much longer than those of KcsA and other Kv channels (43 aa vs. 1423 aa). This long S5-P linker may play a unique role in the structure and function of the HERG channel.
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To explore the role of the S5-P linker in HERG channel function, and to test whether there is a secondary structure in this extracellular domain, we apply cysteine-scanning mutagenesis to positions 571613 of HERG. We also include the P-S6 linker (positions 631638) because, as suggested by the crystal structure of KcsA and by functional studies on the Shaker channel, it interacts with the S5-P linker during gating processes (Doyle et al., 1998; Gandhi et al., 2000
; Larsson and Elinder, 2000
; Ortega-Saenz et al., 2000
). The wild-type (WT) HERG channel has 12 native cysteines that are functionally "silent": channel function is not affected by treatment with DTT, H2O2, or extracellular MTS reagents (Dun et al., 1999a
; Fan et al., 1999
). Therefore, cysteine substitutions are made in the WT HERG background. We examine: (a) channel behavior before and after DTT treatment, and (b) effects of extracellular MTSET or MTSES modification of introduced cysteine side chains on channel function. Based on these data, we propose a structural model for the outer vestibule of the HERG channel, in which the middle part of the S5-P linker assumes an
-helical structure and serves as a bridge of communication between the outer mouth and the voltage-sensing domain. Therefore, the S-P linker in HERG plays a central role in determining the voltage-sensitivity of the C-type inactivation process.
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MATERIALS AND METHODS |
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Oocyte Preparation
Oocytes from Xenopus laevis were isolated as described before (Tseng-Crank et al., 1990), and incubated in an ND96-based medium (in mM: NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5, Na-pyruvate 2.5, pH 7.5, supplemented with 4% horse serum and penicillin/streptomycin) at 16°C. Five to 12 h after isolation, each oocyte was injected with 40 nl of cRNA solution (containing 418 ng cRNA), using a Drummond digital microdispenser. Oocytes were incubated in the above medium at 16°C, and studied 24 d after cRNA injection.
Voltage Clamp Experiments
Membrane currents were recorded from whole oocytes using the "2-cushion pipette" voltage clamp method (Schreibmayer et al., 1994). Both the current-passing and voltage-recording pipettes were filled with 3 M KCl and the tips were plugged with 1% agar in 3 M KCl (tip resistance 0.10.3 M
). During recordings, the oocyte was continuously superfused with a low Cl ND96 solution to reduce interference from endogenous Cl channels (in mM: NaOH 96, KOH 2, CaCl2 1.8, MgSO4 1, HEPES 5, Na-pyruvate 2.5, titrated to pH 7.5 with methanesulfonic acid). The grounding electrodes (containing Ag/AgCl pellets) are filled with 3 M KCl and connected to the bath solution via salt bridges made of 1% agar in 3 M KCl. In some experiments on cysteine-substituted mutants that were poorly expressed, [K+]o was elevated from 2 to 98 mM ([Na+]o removed to maintain the osmolality) to increase current amplitude and facilitate current measurements. This will be specified in the text or figure legends. Voltage clamp was done at room temperature (2426°C) with OC-725B or OC-725C amplifier (Warner Instruments). 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, Inc.). Current data were low-pass filtered at 1 kHz (Frequency Devices) and stored on disks for off-line analysis.
Data Analysis
Most of the voltage clamp protocols and methods of data analysis are described in the text or figure legends. The following software was used for data analysis: pClamp6 or 8, EXCEL (Microsoft), SigmaPlot, SigmaStat, and PeakFit (SPSS).
To explore -helical periodicity in mutation-induced perturbations of channel function, we used a Fourier transform method as described (Cornette et al., 1987
; Li-Smerin et al., 2000
). Assuming that channels are distributed between only two states, closed and open, the free energy needed (
Go) when channels are moved from closed to open states at 0 mV is:
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Cysteine Side Chain Modifications
DTT stock solution (0.5 M in deionized water) was frozen at -20°C in small aliquots. An aliquot was thawed and diluted to 5 mM in bath solution immediately before use. MTSET or MTSES powder (Toronto Research Chemicals) was dissolved in deionized water at 0.1 M shortly before experiments. The stock solution was stored on ice and used within 2 h. After control data were obtained, the MTSET or MTSES stock solution was diluted with bath solution to 1 mM and applied immediately to the oocyte. After treatment with DTT, MTSET, or MTSES, effects on the function of cysteine-substituted channels were evaluated after a 1015 min washout period to ascertain that the effects resulted from covalent modifications.
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RESULTS |
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In the following sections, we will first use data from two mutants (I583C and W585C), along with data from the WT HERG, to illustrate the voltage clamp protocols and data analysis used to characterize the channel phenotypes. We then summarize data from all 48 cysteine mutants. The data allow us to deduce positions in the S5-P and P-S6 linkers where side chain properties are critical for the HERG channel function. Finally, we will focus on two specific issues: (a) periodicity of mutation-induced perturbations of channel function, and (b) effects of MTS modification of pore-entrance residues. These data reveal better defined features in the outer vestibule structure of the HERG channel.
Characterizing I583C and W585C and Comparing them with WT HERG
C-type inactivation and K+ selectivity before and after DTT treatment
In Fig. 2
, data for each of the three channels (WT HERG, I583C, and W585C) are taken from the same oocyte before and after DTT treatment, a reducing agent whose only action is to break disulfide bonds. The voltage clamp protocol is diagrammed on top of column B: from a holding voltage (Vh) of -80 mV, prepulses to 60 mV for 0.2 s are used to fully activate the channels and, if the C-type inactivation is present, to cause channel inactivation. These prepulses are followed by repolarizing steps to Vr ranging from 30 to -120 mV. During the repolarizing steps, channels rapidly ( of a few ms) reach new, lower levels of C-type inactivation determined by Vr. At Vr -40 mV or more negative, recovery from C-type inactivation is followed by deactivation. The tail current (Itail) amplitudes are measured from the plateau level (at Vr positive to -40 mV) or the peak level (at Vr -40 mV or more negative). To pool data from different cells, Itail amplitudes of each cell are normalized by the maximal outward tail current from the same cell. For WT HERG and WT-like mutants (e.g., I583C after DTT treatment, see below), the maximal outward currents occur at Vr -60 mV. For mutants with altered behavior (e.g., I583C before DTT), the maximal outward tail currents are taken from those at Vr 30 mV.
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I583C before DTT treatment has a very different tail I-V relationship from that of WT HERG (Fig. 2 B, a). There is a prominent outward current at 60 mV. Repolarizing steps elicit smaller outward tail currents that reverse at about -20 mV. Therefore, there is a positive slope in the tail I-V relationship in the Vr range of 30 and -60 mV (panel c, open circles). This reflects the change in driving force for current through the channels. Therefore, the C-type inactivation process is disrupted in I583C under these conditions. Furthermore, the Erev (-21.1 ± 6.9 mV) is much more positive than that of WT HERG. Removing extracellular Na+ ions shifts the Erev in the negative direction toward EK, indicating that Na+ ions can permeate through the I583C channels under these conditions. The calculated PK/PNa is 2.3 ± 1.0 (n = 6).
Fig. 2 B, b and c (closed circles), shows that DTT treatment converts the I583C channel phenotype from the mutant behavior to a "WT-like" behavior. DTT-treated I583C shows a prominent negative slope in the tail I-V relationship, indicating a restoration of the C-type inactivation process. DTT treatment also causes a negative shift in Erev (to -93.4 ± 2.6 mV), indicating an increase in PK/PNa (to 117.6 ± 32.4). These observations indicate that the introduced cysteine side chains in I583C are disulfide bonded before DTT treatment. Under these conditions, the channel function (C-type inactivation and K+ selectivity) is disrupted. However, when the cysteine side chains are reduced to the free thiol state, channel function is well maintained.
W585C manifests a mutant behavior similar to that of I583C before DTT treatment: a positive slope in the tail I-V relationship in the Vr range of 30 to -60 mV and an Erev of -23.3 ± 5.5 mV (PK/PNa = 2.1 ± 0.5, n = 5) (Fig. 2 C, a and c). However, unlike I583C, DTT treatment did not convert the mutant behavior to a WT-like behavior (although the W585C current amplitude is modestly increased by DTT treatment; see also of Fig. 5 C, a and b). Relatively aggressive DTT treatment (5 mM for 4 h) cannot convert the W585C phenotype to the WT-like behavior. There are two possible explanations for this apparent resistance of W585C's mutant phenotype to DTT treatment. First, the introduced cysteine side chains in W585C form very stable disulfide bonds that cannot be reduced under our experimental conditions. Second, the tryptophan side chain at 585 is critical for channel function so that replacing it with cysteine, even in the reduced free thiol state, disrupts the C-type inactivation and K+ selectivity.
Effects of MTSET and MTSES before and after DTT treatment
To distinguish between these two possibilities, we test the effects of MTS reagents (MTSET and MTSES) on W585C before and after DTT treatment. MTS reagents can modify free thiol side chains, but not thiol side chains engaged in stable disulfide bonds. Therefore, if DTT-treated W585C has free thiol groups despite the mutant behavior, extracellular MTS should be able to modify the side chains and alter channel function. Furthermore, if there is disulfide formation in W585C before DTT treatment, the MTS effects should be enhanced by DTT treatment.
Figs. 3 A and 4 A show that MTSET or MTSES treatment has no effects on WT HERG. These confirm that native cysteines in HERG are not accessible, or not reactive, to extracellular MTS reagents. Therefore, we are confident that the effects of MTS treatment on cysteine-substituted mutants are due to modification of the introduced cysteine side chains. Fig. 3 B shows that the effect of MTSET on I583C is enhanced by DTT treatment. This is consistent with the notion that the introduced cysteine side chains in I583C are disulfide bonded before DTT treatment. This causes a disruption of channel function (C-type inactivation and K+ selectivity) and a low MTSET sensitivity. Breaking the disulfide bonds by DTT restores the channel function and increases MTSET sensitivity.
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Fig. 4, B and C , illustrate the effects of MTSES on I583C and W585C before and after DTT treatment. Consistent with data shown in Fig. 3, without DTT treatment MTSES has little effects on either channels. After DTT treatment, MTSES markedly suppresses the I583C current amplitude but modestly increases the W585C current amplitude. Therefore, MTSET and MTSES have qualitatively the same effect on I583C, but different effects on W585C. The implication of these observations will be addressed in the context of the proposed structural model for HERG's outer vestibule (see DISCUSSION).
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WT HERG has the same voltage dependence of activation before and after DTT treatment (Fig. 5 A). The activation curve is well described by a single Boltzmann function, with an average half-maximum activation voltage (V0.5) of -17.0 ± 1.5 mV (n = 14). On the other hand, without DTT treatment I583C manifests a more negative Boltzmann component (V0.5 = -40.2 ± 0.9 mV, n = 6, Fig. 5 B, c, open circles) than that of WT HERG. Furthermore, there appears a slowly rising phase of channel activation in the positive voltage range. Therefore, the activation curve requires a second Boltzmann component for a good fit (Fig. 5 legend). DTT treatment of I583C restores the WT-like behavior: the activation curve now follows a single Boltzmann function. The V0.5 value is similar to that of WT HERG (-13.4 ± 2.4 mV, n = 5, Fig. 5 B, c, closed circles). W585C manifests the mutant behavior before and after DTT treatment: its activation curves under both conditions require a double Boltzmann function for a good fit, and the V0.5 values of the negative Boltzmann component are more negative than the V0.5 of WT HERG (-31.5 ± 3.7 mV, n = 5, and -27.4 ± 3.0 mV, n = 7, before and after DTT, respectively).
Summary of Effects of Cysteine Substitutions in the S5-P and P-S6 Linkers on HERG Channel Function, and Effects of MTSET or MTSES Modification
We apply the above voltage clamp protocols and data analysis to the other 46 cysteine-substituted mutant channels made in the S5-P linker (positions 571613) and in the P-S6 linker (positions 631637). Results are summarized in Fig. 6
. Fig. 6 A summarizes "rectification factors" (see equation in Fig. 6 legend), which indicate whether the C-type inactivation process is maintained (values < 0 or upward histogram bars) or interrupted (values > 0 or downward histogram bars). Fig. 6 B summarizes the permeability ratios of K+ to Na+ (PK/PNa). Fig. 6 C summarizes the half-maximum activation voltages (V0.5) of the (major) Boltzmann component used to fit the activation curves (see equation, Fig. 5 legend). Fig. 6, D and E, summarize the ratios of currents after MTSET or MTSES treatment to control (IMTS/IC, as described in Figs. 3 and 4).
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Based on Fig. 6, we can divide the degrees of impact on channel function of side chain modification into three groups: high-impact, intermediate-impact, and low-impact. High-impact positions are those where cysteine substitution disrupts the channel function even when the thiol groups are in the reduced state. This is revealed by a marked MTSET sensitivity with or without DTT treatment, despite the maintained mutant behavior. The three nonfunctional mutants (N573C, K595C, and K638C) are also included in this group. There are a total of 15 high-impact positions: three at the outer end of S5 (I571, G572, and N573), eight in the 583597 segment (W585, L586, N588, L589, G590, D591, I593, and K595), one between the 583597 segment and the P-loop (G604), and three in the P-S6 linker (P632, T634, and K638).
Intermediate-impact positions are those where cysteine substitution in free thiol state is well tolerated (WT-like behavior, some requiring DTT treatment due to disulfide formation), but further MTSET modification produces a significant impact on channel function. There are 10 intermediate-impact positions: seven in the 583597 segment (I583, G584, H587, Q592, G594, P596, and Y597), one between the 583597 segment and the P-loop (D609), and two at the two ends of the P-loop (T613 and S631).
There are 22 positions where introduced cysteine side chains are well tolerated (WT-like phenotype) and do not form disulfide bonds (DTT has no further effects). Importantly, MTSET treatment does not have significant irreversible effects on these cysteine-substituted channels. Since the WT residues at these positions are mostly hydrophilic or even charged, it is likely that the insensitivity to MTSET is not due to a lack of accessibility, but due to a lack of impact of side chain modification on channel function. Therefore, these are low-impact positions where side chains are most likely solvent (extracellular aqueous phase) exposed: 574582, 598603, 606608, 610, 612, 635, and 636.
Finally, there are four positions (P605, Y611, N633, and E637) where cysteine substitution leads to a DTT-resistant mutant phenotype. These cysteine-substituted channels are insensitive to MTSET or MTSES even after aggressive DTT treatment. Therefore, it is unclear whether the introduced thiol side chains are in the reduced state but are not accessible or not reactive to extracellular MTSET or MTSES, or the introduced thiol side chains are in very stable disulfide bonds that cannot be broken by DTT under our experimental conditions. The low expression level of all four mutants suggests that the second possibility may be more likely.
A striking feature of these summary results is the shared common mutant phenotype. Therefore, perturbing the conformation of HERG's outer vestibule, either due to disulfide bond formation involving introduced cysteine side chains (as evidenced by a restoration of WT-like behavior after DTT treatment), or due to a replacement of residues at high-impact positions with cysteine (as evidenced by a prominent MTSET sensitivity after DTT-treatment despite the maintained mutant phenotype), leads to common results: a disruption of C-type inactivation (Fig. 6 A), a decrease in K+ selectivity (Fig. 6 B), and a negative shift in V0.5 of channel activation (Fig. 6 C). These observations support a key role played by HERG's outer vestibule in channel function. Note that the PK/PNa values are rough estimates of channel selectivity, because the intracellular K+ and Na+ concentrations are not directly measured. The estimated PK/PNa values may be mutant-dependent, because mutation-induced changes in membrane conductance may affect these intracellular ion concentrations. However, these uncertainties do not alter the conclusions listed above.
Another striking feature is the marked impact on channel function of cysteine substitution at 15 consecutive positions in the middle of the S5-P linker: the 583597 segment (including 587 studied previously [Dun et al., 1999a]). K595C destroys functional expression. The other 14 channels all manifest the common mutant phenotype before DTT treatment. After DTT, seven assume the WT-like behavior: I583C, G584C, H587C, Q592C, G594C, P596C, and Y597C (rectification factor is switched from positive to negative, PK/PNa is increased, and V0.5 of activation is shifted in the depolarizing direction). The remaining seven maintain the mutant behavior: W585C, L586C, N588C, L589C, G590C, D591C, and I593C. However, the enhancement of their sensitivity to MTSET by DTT treatment (Fig. 6 D) reveals that the introduced cysteine side chains at these positions indeed are reduced, despite the maintained mutant behavior (i.e., high-impact positions).
Periodicity of Mutation-induced Perturbations in Channel-gating Function
The distribution of these seven high-impact positions along the 583597 segment gives a hint of -helical pattern. To further explore this possibility, mutation-induced perturbations in channel activation gating (after DTT treatment) are used to analyze
-helical periodicity as described (Cornette et al., 1987
; Li-Smerin et al., 2000
) (see MATERIALS AND METHODS). Fig. 7 A illustrates the Fourier transform power spectrum of the 583597 segment. For K595C that does not produce functional channels, the value <V> of this segment is used (see MATERIALS AND METHODS; Li-Smerin et al., 2000
). The major peak occurs at an angular frequency of 94°, close to the expected peak of an ideal
-helix (100°). The
-helical tendency, as calculated by
-PI, is 1.64. Although the
-PI is less than two, the level considered as a strong indicator of
-helix formation, it nevertheless supports such a helical tendency. We further analyze the
-helix forming tendency along the S5-P linker from positions 571 to 613, using a 15-aa sliding window (Fig. 7 B). The analysis indicates that the middle part of this linker has the strongest likelihood of forming an
-helix. The amino end and especially the carboxyl end have little or no sign for long
-helices.
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T613 is at the amino end of the P-loop in one-dimensional sequence (Fig. 1). If it is also close to the external entrance of the pore, we predict that MTSET or MTSES modification of T613C should induce a phenotype switch, similar to the effects of MTS modification on S631C. Fig. 8 B shows tail I-V relationships of T613C before and after MTSET or MTSES modification. Before MTS treatment, the tail I-V relationships manifest a prominent negative slope in the Vr range of 30 to -60 mV (strong C-type inactivation) and an Erev close to -100 mV (high PK/PNa). After modification by either MTSET or MTSES, the tail I-V relationships have a positive slope in the same Vr range (C-type inactivation disrupted) and the Erev values are shifted toward 0 mV (PK/PNa reduced). These results confirm our prediction.
Among the other eight positions in the outer vestibule region where cysteine substitution sustains the WT-like behavior and MTS modification has a marked impact on channel function (Fig. 6), only one has the same response as those of S631C and T613C. Fig. 8 A shows that MTSET or MTSES modification of G584C also induces a phenotype switch. This position is 30 amino acids away from the pore entrance in the one-dimensional sequence (Fig. 1). However, the similarity in the MTS effects between G584C and the two pore-entrance residues suggests that in three-dimensional space G584 may be also close to the pore entrance.
Fig. 8 C shows that MTSET and especially MTSES cause a marked suppression of S631C channel conductance. This is not seen in either T613C (Fig. 8 B) or G584C (Fig. 8 A). These observations suggest that 631 side chains may be deeper into the pore than those of 613 or 584. Therefore, in addition to the charge effect on conformational changes around the pore entrance, MTS modification also causes a steric hindrance to current flow through the S631C pore.
Although MTSET modification of D609C also induces a phenotype switch, similar to the effects of MTSET on the pore-entrance residues, MTSES has little effect on D609C (Fig. 8 D). Therefore, the MTSET effects on D609C are due to a mechanism different from an induced electrical repulsion between like charges.
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DISCUSSION |
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The most striking finding in our study is the critical role in the HERG channel function played by the 583597 segment in the middle of the S5-P linker. We propose a structural model (Fig. 9 C) in which the 583597 segment forms an -helix (583597 helix), with its amino end sitting at the pore entrance and the whole helix exposed to the extracellular aqueous environment. This is based on the following observations: (a) analysis of side chain properties suggests that this segment can form an amphipathic
-helix (Fig. 9 B), and
-helix periodicity analysis of mutation-induced perturbations of channel function supports such a secondary structure (Fig. 7), (b) MTS modification of G584C induces a phenotype-switch similar to that seen with MTS modification of two pore-entrance residues (T613C and S631C), whereas none of the other cysteine mutants have such a response, (c) all residues in the 583597 segment are accessible to extracellular MTS reagents (Fig. 6 D), and (d) several resides along this segment (W585, G590, Q592 and I593) are critical for the binding of extracellular peptide toxins, ErgTx (Pardo-Lopez et al., 2002
) and BeKm-1 (unpublished data). Such an
-helix will be
22 Å in length (Fig. 9 B). When oriented parallel to the plane of the cell membrane with its amino end near the pore entrance (Fig. 9 C), its carboxyl end will be able to reach the voltage-sensing domain. This is based on: (a) the dimension of KcsA (Doyle et al., 1998
), and (b) a study using "molecular tapes" to estimate the distance between the pore entrance and the voltage-sensing domain of the Shaker channel (Blaustein et al., 2000
). In this way, this 583597 helix can serve as a bridge of communication between the outer mouth and the voltage-sensing domain. We hypothesize that depolarization-induced motions in the S4 domain (outward and/or rotational movements along with a change in the S4 tilt [Cha et al., 1999
, Glauner et al., 1999
]) can change the orientation of this 583597 helix or push it toward the pore. This then pinches off the external entrance and causes C-type inactivation. Upon membrane repolarization, a reversal of the above S4 movements then relieves the force on the 583597 helix, allowing the pore to reopen (recovery from C-type inactivation). These tightly choreographed molecular motions can account for the high voltage-sensitivity and the rapid onset and recovery kinetics of C-type inactivation in the HERG channel.
In Fig. 9 C, the 583597 helix is depicted as being oriented parallel to the plane of the cell membrane. However, its orientation and interactions with other domains of the channel may change during channel gating (H. Robert Guy, personal communication). It is possible that in some gating state, part of the 583597 helix may become more parallel to the pore axis, with its amino terminus participating in pore lining. This can explain the differential effects of MTS modification of thiol groups introduced to three consecutive positions at the amino terminus of this segment: 583585. As discussed above, the phenotype-switch induced by either MTSET or MTSES modification of G584C can be explained by proposing that residues 584 lie at the pore entrance, with their introduced thiol side chains pointing toward the pore axis. If this part of the helix becomes somewhat parallel to the pore axis, 583 will be deeper into the pore and 585 will be on the channel surface. Increasing 583C side chain volume by either MTSET or MTSES will cause a steric hindrance to current flow through the pore. This can explain why I583C channel conductance is markedly reduced by either MTS reagent (Figs. 3 B and 4 B). On the other hand, adding positive charges to the 585 thiol side chains by MTSET will cause a positive shift in the local surface potential and reduce [K+] around the pore, leading to a decrease in channel conductance. MTSES should have the opposite effects. This may explain why W585C channel conductance is decreased by MTSET but increased by MTSES (Figs. 3 C and 4 C).
Implications of Other High- and Intermediate-impact Positions in the Structure-function Relationship of HERG
High-impact positions at the outer end of S5 and in the P-S6 linker
The crystal structure of a K channel pore domain, KcsA, shows that the outer end of M1 (S5 equivalent) and P-M2 linker (P-S6 equivalent) are close to each other in three-dimensional space (Doyle et al., 1998). Functional studies on the Shaker channel further suggest that the outer end of S5 (E418) interacts with the P-S6 linker (V451/G452) (Fig. 1) (Gandhi et al., 2000
; Larsson and Elinder, 2000
; Ortega-Saenz et al., 2000
). We identify one group of high-impact positions at the outer end of S5 (571573) and another group in the P-S6 linker (632634). It is possible that in the HERG channel, region "571573" interacts with region "632634" through their side chains and peptide backbones during channel gating, and these interactions are important for channel function. Further experimentation is needed to confirm these interactions (e.g., by double cysteine substitution and testing for inter- or intrasubunit disulfide formation involving the introduced thiol groups), and to determine their role in channel function.
Effects of MTS modification of D609C is charge-specific
A string of three charges, two lysines flanking an aspartate (KDK), is located close to the amino end of the P-loop (positions 608610, Fig. 1). Are these charges important for channel function? For example, can they contribute to the voltage-sensitivity of C-type inactivation in the HERG channel? Do they dominate the surface potential around the pore, so that they can influence the pore conductance via effects on the local K+ concentration? Neutralizing any one of these charges (K608C, D609C, or K610C) does not perturb channel function (intact C-type inactivation and strong K+ selectivity). Modification of K608C or K610C by MTSET, and more importantly by MTSES, has little impact on channel function or pore conductance. Fig. 8 D shows that although MTSES has little effect on D609C, MTSET modification induces a phenotype switch: C-type inactivation and K+ selectivity are disrupted. It is possible that the repulsive forces between the positive charge attached to the thiol side chain at 609 by MTSET and the positive charges on the two flanking lysine side chains can limit the backbone flexibility or perturb the conformation to such an extent that the pore function is disrupted. These observations rule out any major role played by these three charged residues in influencing either the voltage-sensitivity of C-type inactivation, or the local surface potential around the pore.
HERG has a "Floppy" Outer Mouth
Conformational flexibility of the outer mouth in the HERG channel is key to its function. In this regard, the reduced hydrogen bonding capability around the outer mouth of the HERG channel, as suggested by the amino acid sequence alignment shown in Fig. 1, is an important contributing factor. In the Shaker channel, oxygen of the carboxylate side chain of E418 can form a hydrogen bond with the peptide backbone nitrogen in the P-S6 linker (451/452) (Larsson and Elinder, 2000; Ortega-Saenz et al., 2000
). The E418 equivalent in the HERG channel, I571, does not have the oxygen donor for hydrogen bonding. In the Shaker, the nitrogen of W434 forms a hydrogen bond with the hydoxyl side chain of Y445 (Doyle et al., 1998
). Again, in HERG the equivalent residues (Y616 and F627) cannot form a hydrogen bond.
In our structural model for HERG's outer mouth, this flexibility allows the 583597 helix to assume different orientations and may become intercalated into the pore lining in some gating state. Therefore, rigidifying the outer vestibule structure by disulfide bond formation involving cysteine side chains introduced here is detrimental to C-type inactivation and K+ selectivity. Cysteine substitutions of critical residues at high-impact positions, or MTS modifications of pore-entrance residues, may also reduce the conformational flexibility and thus produce the same mutant phenotype. In the HERG mutants examined here, a disruption of pore properties (loss of C-type inactivation and K+ selectivity) goes hand-in-hand with a hyperpolarizing shift in the voltage dependence of activation (Fig. 6, AC). This strong association suggests that rigidifying the outer mouth structure makes conformational changes induced by S4 movements propagate to the outer mouth region more efficiently, thus making the HERG channel open more readily. Therefore, we propose that a "floppy" outer mouth in the WT HERG channel makes it more reluctant to open but easier to close (C-type inactivate) than the Shaker channel.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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This study is supported by HL 46451 from the National Heart, Lung, and Blood Institute; National Institutes of Health; and a Grant-in-Aid Award from the American Heart Association/Mid-Atlantic Affiliate (G.N. Tseng).
Submitted: 30 July 2002
Revised: 24 September 2002
Accepted: 30 September 2002
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REFERENCES |
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