Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain
Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4–S5 linker level, but also those split at the intracellular S2–S3 and the extracellular S3–S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2–S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3–S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4–S5 linker, structural integrity of the intracellular S2–S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3–S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.
KeywordsPotassium channel Split channel Gating hERG Voltage sensor
Kv11.1 (hERG, KCNH2) K+ channels mediate the cardiac IKr current that acts as an important determinant of action potential repolarization in the human ventricle and of pacemaking activity in heart nodes [17, 34, 45, 58]. Impairment of Kv11.1 function by mutations in the KCNH2 gene or by a variety of drugs prolongs the QT interval of electrocardiograms leading to inherited and acquired type 2 long QT syndrome, increasing the risk of torsade de pointes arrhythmia, ventricular fibrillation and sudden cardiac death [46, 58]. Furthermore, Kv11.1 is also expressed in a variety of non-cardiac cells in which it plays a key role in setting their electrical behaviour [4, 5, 40, 58].
Kv11.1 belongs to the voltage-gated family of potassium channels (Kv), all of them characterized by a tetrameric molecular architecture in which each subunit contains six transmembrane helices (S1–S6) with a modular organization [7, 52, 68]. In the functional complete channel, the pore domain (PD) is formed by the pore modules (S5–S6 and the intervening pore loop) of all four subunits, which arrange with fourfold symmetry surrounding a unique ion conduction pore. In the assembled tetramer, the PD is also surrounded by four voltage-sensing domains (VSDs) located at the periphery. VSD corresponds to transmembrane helices S1–S4, of which the primary voltage sensitive component is helix S4, containing the positively charged residues that move in response to changes in membrane potential . The concept of Kv channels as a product of a evolutive combination of two functionally autonomous VSD and PD modules [2, 28, 69] is reinforced by (i) the existence of PD-only voltage-independent channels [8, 20, 27, 39] that probably share a common ancestor with Kv and other voltage-dependent channels [23, 29, 69], (ii) the demonstration of functional VSD-only-based voltage-dependent channels [10, 12, 54] and voltage-controlled enzymes in which a VSD resembling those found in voltage-gated channels provides membrane potential control of the catalytic activity  and (iii) the generation of voltage-gated ion channels by either fusing together VSDs and PDs from different sources [3, 33, 53] or co-expressing them as separate protein entities .
In Kv11.1 and other Kv channels, VSD and PD are covalently linked by the so-called S4–S5 linker, classically viewed as a rigid mechanical lever that transmits the voltage sensor reorganizations triggered by changes in transmembrane voltage to the channel gate located at the bottom of transmembrane helix S6 [9, 59]. However, we have recently shown that voltage-dependent Kv11.1 and Kv101.1 functionality can be reconstructed using non-covalently linked VSD and PD (split channels ), pointing to an alternate gating mechanism for this channel diverging from that operating in the more traditionally considered Shaker-like Kv channels [14, 32, 55]. Interestingly, in spite of the very short length of the S4–S5 linker in these channels leading to a non-domain swapped architecture unlike that encountered in the ‘more classical’ Kvs 1–9 [65, 67], some differences in functional outputs can be induced when the split point is moved along the linker. Thus, breaks in the S4 helix/S4–S5 linker connection leads to altered VSD-PD coupling inducing constitutively active Kv10.1 splits  and strongly hyperpolarization-shifted Kv11.1 splits also showing an increased destabilization of closing, a reduced ability to reach more distal closes state(s) and a reduced voltage dependency of both activation and deactivation . On the other hand, interruptions at the C-terminal section of the S4–S5 linker slow down Kv10.1 activation and deactivation kinetics  and give rise to quite normal VSD-PD coupling during activation but markedly accelerated deactivation kinetics in Kv11.1 . Whereas interruptions of the channel protein inside the linker joining the two full-length PD and VSD modules [14, 32, 55], able to act as separate and autonomous functional entities, could be considered a relatively conservative approach, the possibility that a fully functional channel can also be regenerated by combining channel fragments from a protein interrupted at other levels different from the S4–S5 linker has not been evaluated. Therefore, here, we checked this possibility after splitting Kv11.1 at the different loops linking the transmembrane spans of the channel core. Surprisingly, we found that channels covalently interrupted at the intracellular S2–S3 and the extracellular S3–S4 loops, also yield fully functional channel proteins. Careful functional characterization of these constructs suggests that the S2–S3 linker may modulate the transduction of VSD rearrangements to opening and closing of the cytoplasmic channel gate, whereas the covalent continuity of the S3–S4 linker is not essential for proper operation of the Kv11.1 voltage sensing machinery.
Materials and methods
Molecular biology, mutagenesis and expression in Xenopus laevis oocytes
Kv11.1 split channels were generated as PCR fragments containing the desired coding sequences that were inserted into the pSP64A+ vector as HindIII-BamHI fragments. The N-terminal demi-channel fragment for split 478 was synthesized using a sense oligonucleotide containing a HindIII site, a Kozak signal and the sequences for the initial eight Kv11.1 residues together with the corresponding antisense oligonucleotide carrying the coding sequence for residues 466 to 478, followed by a stop codon and the BamHI recognition site. For the C-terminal demi-channel fragment synthesis, the sense oligonucleotide was designed to contain a HindIII site, a Kozak sequence and the start codon followed by the 479 to 489 Kv11.1 coding sequence, whereas the antisense oligonucleotide covered the last 10 residues of the protein (1049–1059) a stop codon and the BamHI recognition sequence. All the rest of Kv11.1 split channels (438, 482, 514, 518, 573 and 637) were generated in an identical manner but to modify the N-terminal of the constructs the antisense oligonucleotide was designed to contain the desired Kv 11.1 final coding sequence before the stop codon. In the case of the C-terminal constructs, the sense oligonucleotide was designed to contain the corresponding coding sequences of the different C-terminal demi-channels after the start codon.
Split channel constructs with the I521C single-point mutation were created by overlapping PCR as previously described [14, 15, 16]. All constructs were analysed by standard fluorescence-based DNA sequencing to confirm the mutations and verify the absence of errors.
Procedures for frog anaesthesia and surgery to obtain oocytes and microinjection have been detailed elsewhere [1, 6, 14, 15, 16, 61]. Oocytes were maintained in OR-2 medium (82.5 mM NaCl, 2 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1 mM Na2HPO4, 10 mM HEPES, at pH 7.5). Cytoplasmic microinjections were performed with 50 nl of in vitro synthesized cRNA per oocyte.
Electrophysiological recording and analysis
Two-electrode voltage clamp recordings were performed as previously described [1, 6, 14, 15, 16, 61] in manually defolliculated oocytes at room temperature 2–3 days after injection, using a Turbo TEC-01C amplifier (NPI electronics). The intracellular electrodes had resistances of 0.4–0.8 MΩ when filled with 3 M KCl. Unless otherwise stated, recordings were obtained in OR-2 medium. In some cases, high-K+ OR-2 medium in which 50 mM KCl replaced an equivalent amount of NaCl was used to maximize currents of those constructs showing a low level of functional expression. Oocytes showing membrane potentials more positive than − 30 mV after impalement with the first electrode in OR-2 medium were discarded. Current recordings were obtained in an experimental chamber of 0.12 ml volume continuously perfused at 2.4 ml/min. For experiments with [2-(trimethylammonium)ethyl]methanethiosulfonate chloride (MTSET; Biotium) the powder reagent was aliquoted, stored at − 20 °C and dissolved in OR-2 just before application to each individual cell. In this case, silver chloride ground electrodes were connected to the bath chamber through agar bridges. Data acquisition and analysis were performed with the Pulse-PulseFit (HEKA Electronics) and IgorPro (WaveMetrics) software packages running on Macintosh computers. Ionic currents sampled at 1 or 10 kHz were obtained using the voltage protocols indicated in the graphs.
The time course of voltage-dependent activation was studied using an indirect envelope-of-tail-currents protocol, varying the duration of depolarization prepulses and following the magnitude of the tail currents on repolarization. The time necessary to reach a half-maximal tail current magnitude was used to compare the speed of activation of the different channels.
Onset of fast inactivation was studied after activation and inactivation of the currents with a prepulse to positive voltages, followed by a second short prepulse to around − 100 mV used to recover the channels from inactivation, and a subsequent test pulse at different voltages to re-inactivate the channels. Time constants for the onset of inactivation were obtained from current traces fitting a single-exponential function to the decaying portion of the currents during the test pulses. The voltage dependence of inactivation was determined with an alternative triple pulse protocol in which cells were depolarized to + 40 mV for several seconds to activate/inactivate the channels and subsequently allowed to relax to a inactivation steady state during a brief test pulse at different voltages, followed by a third step to + 40 mV in which the initial current magnitude was measured to assess the relative number of channels available to activate at the end of the test pulse. Due to the fast deactivation at negative voltages during the test pulses, the closing rates were obtained in each oocyte from biexponential fits to the decaying tail currents as indicated above. These rates were used to determine the proportion of channels closed at the end of every test pulse and to correct for closing-induced decreases in the initial current magnitude at the beginning of the third step .
For experiments designed to study the accessibility of engineered S4 cysteines to MTSET, we routinely used a brief voltage ramp as a stimulatory step that allowed us to kinetically follow the possible shifts in MTSET-induced voltage dependence, either repetitively pulsing the cells at 5-s intervals or maintaining them without pulsing at the indicated holding potentials. All constructs used for this purpose contained a Cys introduced in position 521 of the upper S4 helix (to follow its modification in the presence of MTSET) and two additional mutations (C445V and C449V) to prevent any inadvertent effect caused by MTSET modification of the endogenous cysteines of the Kv11.1 S1–S2 linker. Due to the differences exhibited by different splits in the magnitude of the shift in voltage dependence or in the extent of closing impairment induced by the MTS reagent, the rates and/or the voltage dependence of modification were subsequently obtained quantifying: (i) the magnitude of the peak current increase during the ramp; (ii) the decrease in the time necessary to reach the current peak during the ramp; (iii) the change in the amount of current recorded at the end of a conditioning voltage step to negative voltages (e.g. − 120 mV), included immediately before or after the ramp; and (iv) the decrease in the ratio of the slopes (rectification factor) obtained from the current traces during the steeper raising phase and the minimum slope phase, both in the middle and at the beginning of the ramps, respectively.
Data values given in the text and in figures with error bars represent the mean ± SEM for the number of indicated cells. Comparisons between data groups were at first performed by parametric Student’s impaired t test (two-tailed). When significant differences in standard deviation were present an alternate Welch’s test or non-parametric Wilcoxon or Mann-Whitney test were also used. In all cases, p values < 0.05 were considered as indicative of statistical significance.
Differential effect of interruptions in different loops linking transmembrane helices on Kv11.1 functional expression
Kv11.1 splits interrupted at the extracellular S1–S2 linker (split 438) and at the external loops linking either the S5 helix with the intervening pore loop that surrounds the selectivity filter (split 573), or the pore loop and the N-terminal end of helix S6 (split 637), were not functionally expressed. Surprisingly, robust voltage-dependent Kv11.1-like potassium currents were observed upon co-injection of cRNAs encoding two demi-channels leading to an N-terminal half truncated in the intracellular S2–S3 linker and a C-terminal half covering the rest of the protein up to the final residue 1159 (S2–S3 linker splits) giving rise to fully functional Kv11.1 channel expression. Also, a combination of the N-terminal part of the channel broken at the extracellular S3–S4 linker together with a second half consisting of the remainder of the S3–S4 linker through the C-terminus (S3–S4 linker splits) yielded functional channels. To ensure that the results are not exclusively due to the unique behaviour of a split made at a particular position, in both cases, two different splits were generated in each linker. For the S2–S3 linker, these corresponded to split positions 478 and 482 (Fig. 1). Since the exact positioning and tridimensional appearance of the S3–S4 linker has not been defined in the recently reported cryo-EM structure of Kv11.1 , we also characterized two splits in this loop: split 514 interrupted after the position that marks the middle of the very short S3–S4 linker in the highly homologous Kv10.1 channel (, see upper inset of Fig. 1), and split 518 in which the breaking point is located few residues upstream of position 521 in the Kv11.1 S4 helix that becomes extracellularly extruded from the lipid bilayer when the membrane is depolarized [14, 18]. As previously noticed with Kv11.1 and Kv10.1 channels split at the S4–S5 linker [13, 32, 55], no detectable currents were observed when any of the demi-N or demi-C channel messages alone were injected without their complementary demi-channel counterpart. Indeed, a total absence of functional expression was also obtained when the C-terminal halves of the splits 482 in the S2–S3 linker and 518 in the S3–S4 linker carrying a S620T single mutation in the pore (previously shown to ablate inactivation and substantially enhance Kv11.1 channel expression [26, 61]) were separately injected in the oocyte. This indicates that, providing that the C-terminal half of the channel is expressed on the oocyte surface when injected alone as directly demonstrated with the demi-C portion of Kv10.1 split at the S4–S5 [32, 55], an isolated pore domain is not able to work as a voltage-dependent channel if it only carries a partial section (e.g. the S3 and S4 helices) of the voltage sensor. It also suggests that apposition of complete voltage sensor and pore modules is required to form a complex able to yield a functional, voltage-dependent channel.
Effect of S2–S3 linker interruptions on functional characteristics of the Kv11.1 split channels
Impact of S3–S4 loop interruptions on functional characteristics of the Kv11.1 channels
In this work, we present data that extend our recently reported results, demonstrating that not only a combination of the Kv11.1 VSD and PD modules expressed in Xenopus oocytes as two independent proteins (S4–S5 linker splits, [14, 32]), but also Kv11.1 split channels broken within the VSD at the level of the S2–S3 and S3–S4 linkers, give rise to fully functional channels. The concept of a modular arrangement of Kv channels as a combination of two functionally autonomous VSD and PD modules appears firmly established [2, 7, 28, 52, 68, 69] and could explain the functionality of the S4–S5 linker splits, considering that it is the S4–S5 linker which bridges the voltage sensing and pore domains. However, providing that the two channel halves remain in the oocyte as independent proteins in a single complex, as demonstrated in the case of the highly homologous Kv10.1 channel , it does not easily explain how a functional channel is assembled from two non-covalently joined demi-channels split at other levels outside the S4–S5 linker. In Kv channels, it has been previously shown that a Kv1.2 channel in which the S3–S4 linker has been enzymatically cleaved maintains an almost normal activity . Functional expression in Xenopus oocytes of conducting and non-conducting Shaker channels split at the S3–S4 linker has been also reported , indicating that the structural integrity of this linker is not indispensable for proper synthesis, processing, assembly, quality control and trafficking to the plasma membrane. However, to our knowledge, the successful expression of a S2–S3 linker split Kv channel has not been previously reported. Results of in vitro translation and translocation experiments to understand the membrane topogenesis of the Kv channels have revealed that the membrane insertion of VSD and PD take place independently of each other , but also suggest some differences in the S1 to S4 requirements for integration of different Kv VSDs. Thus, the S3 segment of Kv1.3 and KAT1 does not insert into the membrane by itself [48, 57] and has low membrane insertion activity in KvAP , but that of Shaker efficiently integrates into the membrane . Indeed, in this case, S3 mediates the insertion of the S3–S4 segment in the absence of S2 that acts as an essential factor for optimal integration of this segment in KAT1 [48, 49]. Albeit more complex scenarios are possible, our data with S2–S3 split channels indicate that in Kv11.1 an S2–S3 covalent connection is not necessary for effective insertion of S3, this being consistent with the recognized topogenic properties of segment S2 [48, 49, 57] directing the proper insertion of the demi-N channel half. They would also suggest that either an autonomous S3 or the coordination of S3 and S4 in the S3–S4 segment could also act to insert the demi-C half. Note that even though an intrinsic topogenic activity of S3 exists, the possibility that in the presence of S4 a more efficient membrane integration of S3 takes place  may not be excluded. Regarding helix S4, it has been indicated that in Kv1.3 this segment shows a weaker integration ability than other transmembrane segments , whereas it efficiently inserts into the membrane in the case of KvAP with the help of its signal anchor type I topogenic function . KAT1 S4 does not integrate into the membrane by itself, but it synergistically inserts with S3 in the case of KAT1 and Kv1.3 [48, 49, 57]. Finally, the membrane unstable S4 of Shaker seems to cooperatively act with S3 for fully efficient membrane insertion, and the electrostatic interactions among S2, S3 and S4 play a critical role not only stabilizing the VSD, but also for its optimal membrane integration . Our results with the S3–S4 split suggest that the S4 segment of Kv11.1 does not need the contribution of the initial VSD helices for proper transmembrane insertion. They also demonstrate that in Kv11.1, the presence of S4 and/or any electrostatic pairing with its positive residues is not essential for post-translational membrane integration of S3. If in this case the recognized ability of S4 to insert into the membrane  directs the integration of the C-terminal half of the channel, or if it depends on the signal anchor activity of S5 , remains to be determined. Further work is necessary to determine the relative relevance of each transmembrane segment for the membrane integration of the whole channel structure in this multiple topogenic system.
The study of the functional properties of S2–S3 linker 478 and 482 splits and the comparison with those of the continuous wild-type channel demonstrate that, particularly in the case of the 482 split, some striking analogies exist with the behaviour of the splits interrupted at the C-terminal end of the VSD S4 transmembrane helix (e.g. 539 and 540 splits ). Thus, although a similar half-maximal isochronal activation was found in the 478 split with respect to that of the wild-type channel, a clear shift to more hyperpolarized potentials was exhibited by the 482 split, such as happened with the 539 and 540 splits. A reduction in the amount of equivalent gating charges (z g values) estimated from the slope of the G/V curves and an abolishment of the mode shift behaviour was observed in both S2–S3 linker splits. Furthermore, both constructs showed a clear acceleration of the activation time course in response to membrane depolarization that in the 482 split was more marked and accompanied by a complete absence of the initial delay that precedes the exponential phase of activation typical of Kv11.1. All these properties emphasize the parallelism with the altered gating properties recently observed with channels split at the beginning of the S4–S5 linker . Interestingly, whereas split 478 shows a prominent acceleration of deactivation that more closely resembles that observed in the splits interrupted at the C-terminal end of the S4–S5 linker, this acceleration is slightly reduced in the 482 split, a situation also encountered in those constructs interrupted at the S4 helix/S4–S5 linker connection . The shifts in voltage dependence of current activation were accompanied by similar alterations in the voltage dependence MTSET accessibility to a Cys residue introduced in the upper part of the S4 helix, even though the breaks in the S2–S3 linker do not appear to greatly influence the ability of the S4 segment to dynamically translocate across the membrane in response to depolarizing pulses. Altogether, these data suggest that, albeit less markedly, channels split after residue 482 in the S2–S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 segment.
It is interesting to note that the relatively different behaviour of the 482 split with respect to Split 478, indicates a certain specificity of the S2–S3 linker break position triggering alterations of the kinetic properties, also suggesting that the more marked kinetic modifications observed with the 482 split should not be due to an impairment of the construct ability to assemble in the membrane. On the other hand, the possibility that breaking the linker in other positions may cause even more drastic alterations can’t be excluded. The recently reported cryo-EM structures of Kv11.1 and its highly homologous Kv10.1 indicate that a particularly long S2–S3 linker constitutes a conserved feature of the KCNH family [65, 67] and demonstrate that the most amino terminal region of the channel (the N-tail) lies in close contact not only with the S4–S5 and the C-linkers, but also with the S2–S3 linker . However, the exact role of the S2–S3 linker in Kv11.1 gating remains unknown. We have recently proposed that some interactions between the base of the VSD and the PD in which the N-tail acts as a coupling factor, participate in the modulation of Kv11.1 gating and/or in the voltage-dependent electro-allosteric mechanism that transduces VSD reorganizations to the operation of the PD gate . Given the contacts and central positioning of the N-tail with respect to those regions [14, 65] and the relatively similar impact of N-terminal S4–S5 linker and S2–S3 linker breaks demonstrated here, it is tempting to speculate that the dynamic interplay between the N-tail and all these regions, including the S2–S3 linker, constitutes a crucial component of the Kv11.1 gating machinery.
Our data indicate that the S3–S4 linker interruptions have very little impact on the functional behaviour of Kv11.1. The better exposure to MTSET of Cys 521 in the upper S4 helix can be easily explained if the S3–S4 linker splits allow for a less tightened pathway for the movement of the MTS reagent, this also being consistent with a slightly better access of MTSET in the case of the 518 split, in which the break is located only three residues apart from the cysteine modified at position 521. The solved structure of the S3–S4 linker in Kv10.1 demonstrates that it is constituted by a very short, one amino acid long turn . In this context, our data suggest that the short and probably rigid characteristics of the extracellular S3–S4 linker are not an essential factor for proper working of the voltage sensing machinery. Unfortunately, in the only available tridimensional structure of Kv11.1, the structural organization of the S3–S4 linker is not solved . This also opens the possibility that in this case, a highly disordered and basically flexible region is present in the linker that might not be greatly affected by breaks of the covalent backbone. Therefore, until a more precise structural organization of the S3–S4 linker is available, its exact contribution to Kv11.1 function remains an unanswered question.
The authors thank the expert technical contribution of Teresa González and Dr. Kevin Dalton for proofreading the manuscript.
Pilar de la Peña and Francisco Barros designed the research, performed the experiments and analysed the data. Pedro Domínguez collaborated with some experiments and contributed reagents/materials/analysis tools. Francisco Barros, Pilar de la Peña and Pedro Domínguez wrote the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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