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Received 9 October 1997; accepted after revision 6 May 1998.
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ABSTRACT |
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INTRODUCTION |
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The human ether-à-go-go-related gene (HERG) and the murine ether-à-go-go gene (M-eag) were isolated based on their homology to the Drosophila ether-à-go-go gene (eag; Warmke & Ganetzky, 1994). HERG and M-eag are members of distinct subfamilies within the Eag family which, although only distantly related to the Shaker family of potassium channel genes, encode polypeptides that share important structural features (Warmke et al. 1991). For example, members of the Eag family of potassium channels contain six putative membrane spanning domains, an S4 region shown to mediate voltage-dependent gating in Shaker channels (Papazian et al. 1991; Liman et al. 1991; Logothetis et al. 1992; Schoppa et al. 1992; Bezanilla et al. 1994; Perozo et al. 1994; Zagotta et al. 1994) and a P region highly conserved with those sequences governing ion permeation in Shaker and related channels (MacKinnon & Miller, 1989; MacKinnon et al. 1990; MacKinnon & Yellen, 1990; Yellen et al. 1991; Hartmann et al. 1991; Kavanaugh et al. 1991). Among the superfamily of S4-containing potassium channels, HERG is distinguished by its linkage to a form of congenital long QT syndrome (Curran et al. 1995) and its identification with the native IKr (Sanguinetti et al. 1995; Trudeau et al. 1995), a current important in the repolarization of the cardiac action potential (Sanguinetti & Jurkiewicz, 1990).
Expression studies in Xenopus oocytes indicate that HERG and M-eag channels are both potassium selective and gated by voltage in approximately the same range (Trudeau et al. 1995; Robertson et al. 1996), consistent with a high degree of similarity in their primary structure (49 % in the hydrophobic core). Despite these similarities, M-eag exhibits neither the fast inactivation nor pronounced inward rectification characteristic of HERG and cardiac IKr.
The term 'inward rectification' describes the reduction in outward HERG current amplitudes at positive voltages resulting in a region of negative slope conductance in the I-V relation. Inward rectification is due to a fast inactivation mechanism that limits the occupancy of the open state at positive potentials (Shibasaki, 1987; Sanguinetti et al. 1995; Trudeau et al. 1995; Spector et al. 1996a). During an action potential, therefore, maximal current is achieved only after the membrane begins to repolarize, as channels recover from the inactivated state and pass through the open state prior to closing (Zhou et al. 1998).
The inactivation underlying inward rectification in HERG is similar in many ways to C-type inactivation originally described in Shaker channels (Hoshi et al. 1991). For example, external tetraethylammonium (TEA; Smith et al. 1996) and elevated external potassium (Wang et al. 1996) reduce the rate of inactivation in HERG channels. In addition, inactivation is slowed by a serine-to-alanine mutation in the pore region (S631A; Schönherr & Heinemann, 1996; Suessbrich et al. 1997) and eliminated by a double mutation involving this and one other site in the P region (G628C/S631C; Smith et al. 1996). These results are consistent with the slowing of C-type inactivation in Shaker channels by external TEA (Choi et al. 1991) and potassium (Lopez-Barneo et al. 1993), and by mutations at the equivalent position in the Shaker P region (T449; Lopez-Barneo et al. 1993; Schlief et al. 1996). In contrast to Shaker C-type inactivation, however, inactivation in HERG is inherently voltage dependent (Spector et al. 1996a; Wang et al. 1996). N-type inactivation is not essential for inward rectification, which is maintained when the N terminus is deleted (Schönherr & Heinemann, 1996; Spector et al. 1996a).
We examined the molecular basis of rapid inactivation in HERG using an approach that was independent of pre-existing models. We exploited the structural similarities between HERG and M-eag by creating a library of chimeric channels with an in vivo recombination method in E. coli. We mapped the position of each crossover and recorded currents from the chimeric channels expressed in Xenopus oocytes. By assaying for inward rectification, we mapped the region important for inactivation in HERG channels, identifying critical sites in the upstream P region, the downstream P region and the sixth transmembrane domain (S6). Transplantation of the entire P region and a portion of the S6 domain to M-eag is sufficient to confer inward rectification and inactivation, as well as sensitivity to the class III antiarrhythmic drug E-4031. Moreover, mutagenesis of residues within this region supports previous claims that C-type inactivation mediates inward rectification in HERG.
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METHODS |
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Chimera construction and nomenclature
Chimeras were generated from previously characterized HERG and M-eag clones (HERG: Trudeau et al. 1995; corrected, Trudeau et al. 1996; M-eag: Robertson et al. 1996). The pGH19 vector used was derived from the pGHE expression vector constructed by Liman et al. (1992).
A library of chimeras was produced by in vivo recombination of HERG and M-eag coding regions (Buck & Amara, 1994; Liu et al. 1994; Moore & Blakely, 1994; Levin & Reed, 1995). Parental constructs were cloned in tandem in the same orientation downstream from the SP6 promoter. The tandem was then linearized by digestion at a restriction site between the two coding regions, gel purified and transformed into DH5
cells using heat shock. In vivo recombination events yielded chimeras with single point crossovers in regions of sequence homology between the parental coding regions.
Chimeric channel genes were produced from one of two tandem constructs containing either HERG or M-eag at the 5' ends proximal to the promoter. The crossovers within each chimera were roughly mapped by restriction digests and subsequently pinpointed using fluorescent automated sequencing (ABI). The chimera nomenclature indicates the clone at the 5' end followed by the number of the amino acid residue after which the crossover occurs, followed by the clone at the 3' end. Thus, M(457)H has upstream M-eag sequences, crossing over after M-eag residue 457 to the equivalent position in HERG and extending to the end with HERG sequences. Italics are used for the single letter designation of M-eag and HERG to distinguish them from amino acid residues (e.g. S620). The pore-replacement chimera, containing M-eag sequences except for the P domain and part of S6 from HERG, is designated M (449)H(649)M, or MHM for short.
The pore replacement chimera MHM was constructed from M-eag and chimera H(649)M. Chimera H(649)M was amplified to yield a product encompassing the P region and S6 and thus the crossover point. It also contained an AccI site introduced by means of the 5' primer at a site allowing the in-frame ligation of HERG sequences with M-eag at the homologous site. (A superfluous Acc I site was eliminated with Taq I methylase.) The PCR product and the M-eag clone were each digested with Acc I and Sph I and the appropriate fragments gel purified. The PCR fragment was then subcloned into the corresponding sites in M-eag. Point mutations were generated with the Altered Sites kit (Promega, Madison, WI) or by a two-step PCR reaction in which mutations were introduced into the primers. All sequences exposed to mutagenesis or PCR were confirmed by sequencing both DNA strands using automated fluorescent sequencing.
Oocyte preparation and RNA synthesis and injection
Procedures used for oocyte preparation, injection and recording were similar to those described previously (Rudy & Iverson, 1992; Robertson et al. 1996). Oocytes were collected from female frogs (Xenopus laevis, Nasco) anaesthetized by a 10-20 min exposure to 3-aminobenzoic acid ethyl ester (MS222; 0·5 g in 500 ml water). A small incision (1 cm) was made on one side of the abdomen over the ovary, first in the skin and then in the abdominal muscle layer. The exposed ovary, containing the oocytes, was then exteriorized with blunt forceps. Several lobes of oocytes were removed from the ovary with a single cut using fine scissors. The remaining ovarian tissue was replaced in the abdomen, which was closed, by suturing first the muscle layer and then the skin. Frogs were allowed to recover from the anaesthetic in shallow water. All procedures were in accordance with the animal care guidelines of the NIH and the University of Wisconsin Research Animal Resource Center. Oocytes were defolliculated by hand or with an osmotic shock procedure (Pajor et al. 1992). Transcripts were synthesized from the T7 promoter of linearized DNA templates at the Not I site using the mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA). For injection, the resulting cRNA was diluted to 50 nl in sterile water to a concentration sufficient to yield 0·5-10 µA maximal outward current under voltage clamp. Oocytes were maintained for 1-10 days in ND96 ((mM): 96 NaCl, 2 KCl, 1 MgCl2, 1·8 CaCl2, 5 Hepes, adjusted to pH 7·4 with NaOH and supplemented with 10 µg ml-1 gentamicin sulphate).
Electrophysiological measurements
Currents were recorded using the two-electrode voltage clamp method (OC-726C, Warner Equipment Corporation, Hamden, CT, USA). pCLAMP 6.0 software (Axon Instruments) was used for data acquisition and analysis. Electrode resistances were 0·5-1 M
when filled with 2 M KCl. The bath solution contained (mM): 95 NaCl, 5 KCl, 1 MgCl2, 0·3 CaCl2, 5 Hepes, adjusted to pH 7·4 with NaOH. In some cases, small corrections for leak were made using a linear leak subtraction protocol (based on current evoked at voltage steps to -100 mV) but correction was often not necessary due to negligible leak (< 1 % of total conductance). If leak exceeded 10 % of total conductance the data were discarded. Sample numbers (n) refer to the number of individual oocytes recorded. E-4031 was a gift from Eisai Ltd, Ibaraki, Japan.
The steady-state I-V relationships of HERG chimeras and point mutants were generated by normalizing outward current at the end of the 1 s pulse (which reflects the equilibrium of the open and inactivated states at the various voltages) to the maximal current evoked by a step to 60 mV using a three-pulse protocol (which reflects maximal current with most inactivation removed; see Fig. 4C, inset). For M-eag, currents were normalized to the maximal current evoked at the end of the 1 s voltage command to 60 mV. For MHM, currents were normalized to the peak transient current. Inactivation rates were also determined using the three-pulse protocol, as shown in Figs 4 and 8.
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RESULTS |
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Parental phenotypes
Two-electrode voltage-clamp recordings from parental HERG and M-eag channels expressed in Xenopus oocytes illustrate the key differences in their physiological properties (Fig. 1). HERG currents activate slowly and exhibit a gradual decrease in outward current amplitude at increasingly positive voltages due to rapid inactivation (Fig. 1A; Sanguinetti et al. 1995; Trudeau et al. 1995). Upon repolarization, a hook-like tail current is evoked as inactivation recovers and the channels pass through the open state prior to closing (arrow, Fig. 1A). The tail currents are large in relation to the inactivated outward currents. M-eag currents activate more rapidly than HERG currents and exhibit no apparent inactivation (Fig. 1B). M-eag tail currents decay rapidly and appear small in relation to the large outward currents. Inactivation in HERG is manifested in the I-V relation as a region of negative slope conductance, or inward rectification (arrow, Fig. 1C). The I-V relation of M-eag increases monotonically, with a slight saturation at higher voltages (Fig. 1C). The slope characteristics of the I-V relationship were the primary assay for inward rectification in the mapping experiments described below.
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A, HERG, and B, M-eag currents elicited by 1 s pulses from -80 to 70 mV in 10 mV increments, followed by repolarization to -100 mV, as shown below traces in B. For clarity, only every other trace is shown. The holding potential was -80 mV. C, current-voltage relation for HERG (n = 7) and M-eag (n = 4) based on the protocol shown in B. Points are means ± | ||
Mapping the borders for inactivation
Chimeric constructs and point mutants were used to identify regions responsible for the pronounced inward rectification that distinguishes HERG from M-eag channels. This study focused on chimeras with crossover points in and around the putative pore region, as depicted in Fig. 2. M-eag sequences are represented as open regions and HERG sequences as filled regions, with points of crossover indicated by labelled, vertical lines.
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Alignment of HERG and M-eag amino acid sequences, with dashes representing residues in M-eag which are identical to the aligned residues in HERG. Graphically depicted below this are HERG sequences as filled areas and M-eag sequences as open areas. Chimeras contain regions of both parental channels, crossing over at homologous sites labelled a-d below. See Methods for chimera nomenclature. Point mutation sites in HERG are labelled above the residues mutated. | ||
Mapping the upstream border for inactivation was facilitated by two chimeras with M-eag 5' ends, M (459)H and M (457)H (see Methods for explanation of chimera nomenclature). Chimera M (459)H, containing M-eag sequences to crossover point c in the P domain, exhibits no inactivation and the more linear I-V relation characteristic of M-eag (Fig. 3A and C). In contrast, chimera M (457)H, with a crossover just two amino acids upstream at point b, exhibits diminishing current amplitudes at higher voltages (Fig. 3B), manifested as a region of negative slope conductance in the I-V relation (Fig. 3C). These results suggest that chimera M (457)H possesses key elements of the HERG machinery responsible for inactivation and that the difference between the two chimeras, the phenylalanine at HERG residue 619 (F619) and the serine at residue 620 (S620), lying between crossover points b and c (cf. Fig. 2), may be important determinants of inactivation.
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A, family of currents from chimera M (459)H showing an M-eag-like phenotype with only slight saturation at higher voltages. B, chimera M (457)H, which differs from M (459)H by only two residues, shows a progressive reduction in current amplitude with increasing voltage above 30 mV (n = 3). Cartoons in A and B show approximate crossover points, with labels (b and c) corresponding to exact crossover points in Fig. 2. C, current-voltage relations for the chimeras compared with the parental HERG and M-eag illustrate the substantial negative slope conductance in chimera M (457)H. Points represent means ± | ||
We mutated HERG at S620 to the corresponding residue in M-eag to test the importance of this site in inactivation (Fig. 4). The resulting S620T mutant exhibits little or no inactivation (Fig. 4A) and a corresponding loss of inward rectification in the I-V relation (Fig. 4B). Accordingly, inactivation measured with the three-pulse protocol is slowed to such an extent that it cannot be fitted on the time scale used (Fig. 4C). The dramatic phenotype of S620T is interesting considering that this substitution results in the addition of only a single methyl group. The results from these chimeras and point mutations together indicate that S620 plays a role in inactivation in HERG and represents the upstream border of the region required for inactivation.
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A, currents from HERG point mutant S620T exhibit little inactivation or inward rectification. B, current- voltage relations for S620T (n = 4) compared with HERG and M-eag. Points are means ± | ||
In mapping the downstream border for inactivation, we utilized a chimera with a HERG 5' end. Chimera H (650)M, which crosses over at point d upstream in the mid-S6 region, retains the rapid inactivation and inward rectification of HERG (Fig. 5A and B). Chimera H (650)M differs from HERG in its voltage dependence of activation (g-V curve, not shown), which is left-shifted by about -40 mV, resulting in faster activation kinetics, an early peak of outward current and a more negative peak of the I-V relation. The similarities in the inactivation phenotype nevertheless indicate that the sequences responsible for inactivation lie upstream from HERG residue 651. As will be shown below, one residue critical for inactivation lies within S6 just seven residues upstream from this crossover point.
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A, family of currents from chimera H (650)M, crossing over in mid-S6 at crossover point d. B, current- voltage relation indicates pronounced negative slope conductance and inward rectification for this chimera (means ± | ||
Transfer of rapid inactivation from HERG to M-eag
These results suggest the presence of a discrete inactivation domain responsible for inward rectification in HERG. This region includes much of the P region and S6 transmembrane domain. We tested this hypothesis by transplanting the P region and part of S6 from HERG into M-eag (chimera M (449)H (649)M, or MHM), and found that M-eag was converted into a rapidly inactivating channel with the steep negative slope conductance characteristic of HERG (Fig. 6A and B). Interestingly, this chimera forced the segregation of other HERG gating characteristics. For example, the chimera retains the rapid activation of M-eag, resulting in a transient outward current reminiscent of the Shaker current phenotype (Fig. 6C). In addition, despite significant inward rectification, the tail currents are very fast and lack the rising phase or 'hook' attributed to recovery from inactivation in HERG. Despite these differences, the MHM chimera demonstrates that the differences in primary sequence conferring inward rectification to HERG can be localized to a finite region that includes much of the pore. The voltage sensitivity of inactivation, though shifted by approximately 30 mV, is preserved (Fig. 6D), indicating that the voltage sensor for inactivation is also contained within the replacement region or is conserved between the two channel proteins.
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A, currents elicited from chimera M (449)H (649)M (or MHM) show inactivation. B, pronounced negative slope conductance of the MHM chimeric channels (n = 6). The greater area under the curve reflects the faster activation and slower inactivation of MHM relative to HERG, resulting in reduced inward rectification. C, Shaker-like transients are apparent in MHM at depolarized voltages (40 mV in this trace) when viewed on a faster time scale. D, voltage dependence of inactivation in HERG (n = 6) and MHM channels (n = 8). For HERG, time constants ( | ||
Transfer of E-4031 sensitivity from HERG to M-eag
Previous evidence suggests that class III antiarrhythmic drugs such as E-4031 exert their inhibitory effects by binding internally to the open HERG channel (Kiehn et al. 1996; Spector et al. 1996b). In contrast to HERG channels (Fig. 7A), M-eag channels are insensitive to E-4031 (Fig. 7B). We determined whether the putative pore region transplanted from HERG into M-eag in the MHM chimera contained enough of the drug binding site to confer sensitivity to E-4031. Not only is the MHM channel sensitive to E-4031, but its range of sensitivity is the same as that of the wild-type HERG channel (Fig. 7C and D; IC50 = 348 ± 34 nM for wild-type; 346 ± 67 nM for MHM chimera; n = 4 for each). Thus, the HERG sequences required for inactivation are also sufficient to induce E-4031 sensitivity in channels otherwise containing M-eag sequence.
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A, block of HERG channels by the methanesulphonanilide E-4031. B, M-eag channels are relatively insensitive to the drug. C, pore-replacement chimera MHM, like HERG, is blocked by E-4031. D, dose-response curves give IC50 values for HERG (347·64 ± 34·13, n = 4) and chimera MHM (346·06 ± 66·90, n = 4; n = 5 for M-eag). Steady-state block was achieved by equilibration in the presence of the drug (100 nM, 500 nM, 1 µM, 5 µM) by holding at -35 mV for 10 min (Spector et al. 1996b). Oocytes were then returned to a holding potential of -80 mV and 10-30 s later a single pulse to 20 mV for 1 s was delivered. Block was determined as the reduction of the current relative to control at the end of this pulse. A dose-response curve was fitted using y = 1/[1 + ([E-4031]/IC50)p] where p = Hill coefficient. | ||
Homology with C-type inactivation in Shaker
Our chimera studies suggest that the inactivation machinery resides in the pore region, consistent with previous suggestions of a C-type inactivation mechanism (Schönherr & Heinemann, 1996; Smith et al. 1996). However, the similarity between inactivation in HERG and C-type inactivation in Shaker has recently been challenged based on functional differences between the two processes (Wang et al. 1997). For example, inactivation in HERG is inherently voltage dependent (Spector et al. 1996a; Wang et al. 1996), whereas C-type inactivation in Shaker is not (Hoshi et al. 1991).
Despite these differences, we have observed a remarkable conservation of function at two sites previously associated with C-type inactivation in Shaker channels, one in the P region and another in the S6 putative transmembrane domain. The most extreme phenotype results from mutation of HERG P region residue S631 to a valine (S631V), which eliminates or dramatically slows inactivation (Fig. 8A). The mutant tail currents begin to relax immediately upon repolarization, as if the channels were simply deactivating (closing); the 'hook' attributed to recovery from inactivation in wild-type channels is absent (Fig. 8B). The steady-state I-V curve lacks the negative slope conductance characteristic of wild-type HERG currents (Fig. 8B). This phenotype is reminiscent of the effect of homologous Shaker mutation T449V at the outer mouth of the pore, which eliminates C-type inactivation (Lopez-Barneo et al. 1993).
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A, family of traces from HERG mutant S631V lack inactivation. B, tail currents from HERG wild-type (taken from Fig. 1A) and S631V (from A) superimposed and scaled to the peak amplitudes to show the loss of the 'hook' attributed to recovery from inactivation. Tails were evoked at -100 mV subsequent to a 60 mV step. The dotted line represents the zero current level. C, comparison of current-voltage relations of the S631V mutant and HERG wild-type illustrates the loss of negative slope conductance in the mutant (n = 5). D, comparison of inactivation rates of HERG and point mutants S631V, S631A and V644A at 60 mV. Inactivation currents were elicited using the 3-pulse voltage protocol shown in the inset to Fig. 4C . For the S631V, S631A and V644A mutants, the duration of the hyperpolarizing pulse was shortened to 10 ms and to 15 ms for V644A due to the absence of an inactivation recovery phase or faster recovery rates in these mutants. Inactivation time constants were as follows (means ± | ||
A comparison of relative inactivation rates attributable to mutations in the P region and in S6 underscores the similarities with the homologous Shaker mutants. For example, the HERG inactivation rate is fastest in the wild-type construct (S631), with the rates decreasing in the order S631 > S631A > S631V for mutants in the P region (Fig. 8D and E). This sequence is comparable to the Shaker inactivation rates of mutants at the homologous site, T449S > T449A > T449V (Lopez-Barneo et al. 1993; Schlief et al. 1996). Thus, as in Shaker channels, the HERG inactivation rate increases with the hydrophilicity of the residue at site 631. At a second site in the S6 domain, the relative inactivation rates of HERG wild-type and a mutant decrease in the order V644 > V644A (Fig. 8D and E), and are comparable to the relative inactivation rates of homologous Shaker constructs A463V > A463. These results suggest that HERG channels have a C-type inactivation mechanism that depends on structures homologous to those originally described for Shaker channels.
Our results are consistent with a single inactivation process that is affected by mutations at S631 in the downstream P region, V644 in S6, and S620 in the upstream P region. Evidence supporting this hypothesis includes (1) the single exponential fit to the inactivation currents and (2) the nearly null inactivation phenotypes exhibited by S620T and S631V, rather than the partial, additive effect expected if they independently altered two separate processes.
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DISCUSSION |
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In this study we employed chimeric analysis and point mutagenesis to identify a domain critical for the inward rectification and E-4031 drug sensitivity which distinguish HERG from M-eag. This domain, containing the P region and about half of S6, confers rapid inactivation, inward rectification and E-4031 sensitivity upon M-eag when transplanted from HERG. The properties of the MHM pore-replacement chimera indicate that the residues specifying rapid inactivation and E-4031 binding in HERG are present within a discrete, contiguous domain. This does not exclude other domains of the protein from participating in inactivation or drug binding, but rather constrains such domains to be conserved between the two proteins.
Other unique elements of HERG outside the pore replacement region may also be involved in inactivation because the inactivation rate is somewhat slower and inward rectification is less extreme in MHM compared with the corresponding properties of the parental HERG channels. In addition, although the MHM chimeric channels exhibit significant inactivation, the fast, small tails are reminiscent of M-eag deactivation. One possible explanation for the small tails is that deactivation is faster than recovery from inactivation in MHM, limiting the time spent in the open state and effectively 'clipping' the tail currents. Alternatively, the small tail currents in MHM channels could arise if recovery from inactivation were via the closed state, in contrast to HERG channels, which recover via the open state. Whatever the mechanism, the fast deactivation and small tails may be determined by regions of M-eag sequence in MHM. The involvement of downstream M-eag residues can be ruled out because chimera H(650)M, which crosses over at the same S6 position as MHM, exhibits large, slow tail currents. The potential involvement of upstream M-eag residues, particularly in the N terminus, is consistent with previous studies showing regulation of deactivation rate by the N terminus in HERG channels (Schönherr & Heinemann, 1996; Spector et al. 1996a) and in isoforms of the HERG homologue Merg (London et al. 1997).
Previous descriptions of C-type inactivation in Shaker have identified critical residues at the sites homologous to S631 and V644 in the HERG polypeptide. We show here that parallel substitutions of residues at these sites in the two channels confer similar phenotypes, as reflected in the relative rates of inactivation. These observations reveal a remarkable degree of functional similarity and strong selective pressure for C-type inactivation despite the extensive divergence in other structural features since the time these channels evolved from a common ancestral gene. The marked differences in inactivation between M-eag and HERG argue against the conservation of C-type inactivation as a secondary consequence of selective pressures on the highly conserved conduction pathway.
Our results reveal two mutations with profound effects on inward rectification in HERG. When present individually, S631V and S620T eliminate the negative slope conductance of the I-V plot and dramatically slow or remove inactivation. Since each of these mutations confers a null, or near-null, phenotype, they probably regulate a single process or multiple processes that are tightly coupled. S620T and S631V each slow the measured inactivation process to such an extreme as to rule out additive effects of two independent (non-coupled) processes. This result argues against previous suggestions that inactivation in HERG may also involve a P-type mechanism (Wang et al. 1997), despite the implication of a residue at the equivalent position to S620 in P-type inactivation of Kv2.1 channels (De Biasi et al. 1993). Because P-type inactivation in Kv2.1 exhibits a slow recovery and little voltage dependence (De Biasi et al. 1993), we find little rationale for postulating such a mechanism, while acknowledging that several aspects of inactivation in HERG are not well understood. For example, the voltage dependence of inactivation has yet to be explained at the mechanistic level, but it seems plausible that the inactivation machinery could be similar to that in Shaker channels yet involve the movement of charged residues partly within the electric field. Moreover, the effects of S620T on inactivation in HERG are consistent with the finding in Shaker channels that C-type inactivation involves movements deep within the pore (Molina et al. 1997) where, based on structural models for Shaker channels (Durell & Guy, 1996), S620 is expected to reside.
Consistent with the findings for Shaker inactivation, the HERG inactivation rate is correlated with the hydrophilicity of the residue at site 631 in the downstream pore region, as if the inactivated state is stabilized by hydrophilic residues at that site and destabilized by hydrophobic residues. In the upstream pore region, addition of only a methyl group to the site (S620T) prevents inactivation, as if by sterically hindering essential movements of the polypeptide at that site. At site 644 in S6, in contrast, the larger residue confers the faster inactivation rate (V > A). Since single mutations at either S620 or S631 exhibit essentially null inactivation phenotypes, each site is critical for the stability of the inactivated state. One of the mutations we describe here as giving an intermediate inactivation phenotype, S631A, was previously described as a null (Schönherr & Heinemann, 1996), but later studies by the same authors demonstrated an intermediate inactivation phenotype in agreement with our results (Suessbrich et al. 1997). The range of intermediate phenotypes depends on the residue present and can be predicted by the consequences of homologous mutations in Shaker, providing confirmatory evidence that a highly conserved C-type inactivation underlies inward rectification in HERG.
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Acknowledgements
We thank Jinling Wang for many helpful discussions, Richard Schell, Tuan Thai and Angelina Zappia for their technical assistance and Ebru Aydar, Janet Branchaw, Cindy Czajkowski, Meyer Jackson, Craig January, Jinling Wang and Zhengfeng Zhou for their comments on the manuscript. We thank E. Goulding and S. Siegelbaum for the pGH19 expression vector. This work was supported by NIH grant R01-HL55973-01, a grant from the HHMI-UW Medical School Faculty Development Program, and American Heart Association, Wisconsin Affiliate postdoctoral (I. M. H.) and predoctoral (M. C. T.) fellowships.
Corresponding author
G. A. Robertson: Department of Physiology, 129 S.M.I., University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI 53706, USA.
Email: rbertson{at}macc.wisc.edu
Author's present address
I. M. Herzberg: Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109-0632, USA.
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to the inactivating current evoked at 40 mV, was 3·3 ± 0·2 ms (mean ±