KCNQ1 Assembly and Function Is Blocked by Long-QT Syndrome Mutations That Disrupt Interaction With Calmodulin
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Smita Ghosh, Deborah A. Nunziato, Geoffr
参见附件。
the Departments of Pharmacology (S.G., D.A.N., G.S.P.) and Medicine, Division of Cardiology (G.S.P.), College of Physicians and Surgeons of Columbia University, New York, NY.
Abstract
Calmodulin (CaM) has been recognized as an obligate subunit for many ion channels in which its function has not been clearly established. Because channel subunits associate early during channel biosynthesis, CaM may provide a mechanism for Ca2+-dependent regulation of channel formation. Here we show that CaM is a constitutive component of KCNQ1 K+ channels, the most commonly mutated long-QT syndrome (LQTS) locus. CaM not only acts as a regulator of channel gating, relieving inactivation in a Ca2+-dependent manner, but it also contributes to control of channel assembly. Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus. This CaM-regulated process is essential: LQTS mutants that disrupt CaM interaction prevent functional assembly of channels in a dominant-negative manner. These findings offer a new mechanism for LQTS defects and provide a basis for understanding novel ways that intracellular Ca2+ and CaM regulate ion channels.
Key Words: KCNQ1 KvLQT1 IKs calmodulin long-QT syndrome
Introduction
The long-QT syndrome (LQTS) is a collection of inherited and acquired arrhythmogenic diseases characterized on ECG by a prolonged QT interval. This ECG manifestation reflects an abnormally prolonged ventricular action potential, which can be the substrate for life-threatening arrhythmias that lead to syncope or sudden cardiac death. LQTS is a "channelopathy;"all except one LQTS loci reside in genes encoding ion channels that regulate the cardiac action potential, and characterization of LQTS mutations in specific channels reveals defects that promote excessive depolarization or weakened repolarization.1–3 Nevertheless, development of arrhythmias is rare, and the specific triggers that induce them are not well understood.
Intracellular Ca2+ is one factor with a large potential impact that has only recently been explored.3–7 As Ca2+ is the fundamental second messenger of electrical activity in excitable cells, it is not surprising that many ion channels are subject to feedback modulation by resultant Ca2+ fluxes, a process often mediated by the ubiquitous Ca2+-binding protein calmodulin (CaM).8–13 The recent identification of two possible CaM interaction sites within the C-terminus of KCNQ1,14 the most commonly mutated locus (LQT1) in congenital LQTS,15 offered a window into how Ca2+ may contribute to LQTS. As the pore-forming subunit of the catecholamine-sensitive cardiac IKs current that determines action potential duration on a beat-to-beat basis,16,17 KCNQ1 is well positioned to integrate changes in intracellular Ca2+ into an alteration in action potential duration, consistent with previous reports showing that IKs is a Ca2+-responsive current.18–20 Moreover, the neuronal homologs KCNQ2 and KCNQ3 form heteromultimers that are responsible for the Ca2+-sensitive M current21 in the brain and have also been shown to bind CaM,14,22 suggesting that Ca2+/CaM could be a general regulator for the KCNQ K+ channel family. Nevertheless, attempts to define whether and how Ca2+/CaM regulates any of the KCNQ homologs have produced conflicting results.22–24 Moreover, KCNQ1 stood apart in these initial studies because Ca2+/CaM did not appear to regulate KCNQ1 or IKs currents in a heterologous system and because CaM was not found to interact with KCNQ1. Here, we report that CaM binds to the KCNQ1 C-terminus, where it contributes to channel assembly. On mature channels, CaM appears to relieve channel inactivation in the presence of basal levels of intracellular Ca2+. Further relief of inactivation may augment KCNQ1 current when Ca2+ is elevated.
Materials and Methods
Additional Materials and Methods are available in the online-only data supplement at http://circres.ahajournals.org.
Molecular Biology
Bacterial and oocyte expression constructs were generated with standard approaches. Cloning of the KCNQ1 concatemer yielded Gly-Ser dipeptide between the 2 KCNQ1 repeats.
Electrophysiology
Two electrode voltage-clamp recordings from Xenopus oocytes were preformed as previously described25 with the use of ND-96 bath solution. Intracellular Ca2+ manipulations were performed by micro-injection of 50 to 100 nL of 100 mmol/L BAPTA, 100 mmol/L EGTA, 100 μmol/L CaCl2, or 500 mmol/L sucrose (in 10 mmol/L HEPES, pH 7.4).
Protein Expression and Purification
The KCNQ1 CT constructs, with and without CaM or CaM1234, were isolated from BL-21 (DE3) cells and purified with Talon metal affinity resin (Clontech). Gel filtration was performed over a Superdex 200 HR 10/30 column on an AKTA FPLC (Amersham Biosciences) in 500 mmol/L NaCl, 20 mmol/L Tris, pH 7.5, and 10 μmol/L CaCl2.
Chemical Cross-Linking
Disuccinimidyl glutarate 2.5 mmol/L was added for 5 minutes at room temperature to the purified KCNQ1 CT/CaM complexes before the addition of SDS-PAGE sample buffer.
Statistical Analysis
Data are presented as mean±SEM. Significant differences were assessed using Student t test at P<0.05.
Results
CaM Interacts Directly With the KCNQ1 C-Terminus
To directly assess whether CaM interacted with the KCNQ1 CT, we used paradigms successful in defining CaM interaction with other voltage-gated ion channels.6,26 We first attempted to generate recombinant protein under detergent-free conditions for quantitative binding assays with a 6xHis-tagged KCNQ1 construct of the entire CT (aa 354–676; Figure 1A), but despite abundant protein expression, none was soluble (Figure 1B). This result was similar to that observed for the CaM-binding regions in the CTs of Cav1.2, Nav1.2, and Nav1.5 channels, so we coexpressed CaM with the KCNQ1 CT and found that the KCNQ1 CT became soluble (Figure 1B). Not only did CaM promote KCNQ1 CT solubility, but it was also a KCNQ1 CT binding partner, as indicated by CaM copurification with the 6xHis-tagged KCNQ1 CT by metal affinity chromatography (MAC) (Figure 1B). This interaction was specific, as CaM was not purified in the absence of KCNQ1 CT coexpression (not shown). Further, this interaction could be Ca2+-independent, as CaM1234, a CaM mutant deficient in Ca2+ binding,9 also copurified when coexpressed with the KCNQ1 CT (Figure 1B; see below). The interaction also was Mg2+-independent, as no Mg2+ was added to the buffers. Although consistent with previous findings that CaM interacts with the CTs of other KCNQ family members,14,22,24 this was the first direct demonstration that CaM interacted with KCNQ1.
The Stoichiometry Between CaM and the KCNQ1 CT is 1:1
Although KCNQ CTs contain two potential CaM binding sites (IQ1 and IQ2 in Figure 1A),14 the stoichiometry between CaM and any KCNQ channel has not been determined. As a first estimate, we evaluated the purified KCNQ1 CT/CaM complex on size exclusion chromatography. The complex ran as a single species with an apparent Mw significantly greater than the sum of the 6xHis-tagged KCNQ1 CT and CaM (Figure 2A). The elution profile for the complex was unchanged whether saturating Ca2+ (1 mmol/L) or EGTA (5 mmol/L) was added to the column buffer, further supporting the hypothesis that the interaction between CaM and the KCNQ1 CT did not require Ca2+ (but see below). These data, however, did not yield an accurate assessment of the complex’s Mw or the stoichiometry between CaM and KCNQ1 CT. We therefore performed chemical cross-linking with disuccinimidyl glutarate (DSG), a homobifunctional, non-cleavable reagent reactive toward free amino groups, and subsequent polyacrylamide gel electrophoresis. After 5 minutes’ exposure to DSG at 25°C, none of the 36 kDa KCNQ1 CT input material remained; instead, a single predominant band migrating at 240 kDa was visible after Coomassie staining (Figure 2B, left). CaM was not visible in the input material (lane 1), having migrated off the 7% polyacrylamide gel (arrow depicts the gel’s dye-front), but it could be readily detected in the higher Mw cross-linked material by immunoblot (Figure 2B, right). Similar results were obtained with the KCNQ1 CT/CaM1234 complex (Figure 2B, right). The apparent Mw of the complex, which included both CaM and the KCNQ1 C-terminus, was less than 250 kDa on SDS-PAGE and was therefore consistent with a 4:4 (KCNQ1 CT:CaM) heteromultimer (estimated Mw of 212 kDa), rather than a 4:8 heteromultimer (estimated Mw of 280 kDa). This was consistent with one CaM molecule bound for each putative CaM binding site.
To establish whether one CaM molecule per KCNQ1 CT was sufficient, we fixed the stoichiometry at 1:1 by expressing a construct in which CaM or CaM1234 was joined by a 12x glycine (G12) linker at the KCNQ1 construct’s CT (CT-G12-CaM or CT-G12-CaM1234). These constructs were soluble without the coexpression of unlinked CaM (Figure 2C), and DSG cross-linking of the purified material yielded a product that migrated at 4x the Mw of the untreated material (Figure 2D). The concatemers alo allowed us to determine relative affinities of CaM and CaM1234 for the KCNQ1 CT. When coexpressed with the CT-G12-CaM construct, unlinked CaM, but not unlinked CaM1234, co-purified on MAC (Figure 2E, arrow). This suggested that unlinked CaM but not CaM1234, could complete with linked CaM for association with the KCNQ1 CT. Thus, although CaM could bind to the KCNQ1 CT in the absence of Ca2+, Ca2+/CaM had a higher relative affinity.
We used the in vitro formation of the KCNQ1 CT tetramer to test definitively whether amino acids 589 to 620 were necessary for assembly, as this region had been previously identified as the KCNQ1 assembly domain,27 but a recent study suggested instead that it functioned as a trafficking signal.28 We generated a KCNQ1 CT truncated after amino acid 555 (555, Mw 24 kDa) to exclude 589 to 620. As with the full length KCNQ1 CT, the 555 required CaM coexpression to generate soluble material suitable for MAC purification (not shown). Chemical cross-linking with DSG generated products of 45 kDa, consistent with the inclusion of one 555 KCNQ1 CT and one CaM molecule (Figure 2F, single arrowhead); 100 kDa, consistent with a dimer of the 555 KCNQ1 CT/CaM complex (Figure 2F, double arrowhead); and 200 kDa, consistent with a tetramer of complexes (Figure 2F, asterisk). These results suggested that the KCNQ1 CT/CaM complexes were able to assemble independent of amino acids 589 to 620.
LQTS Mutations in Either IQ1 or IQ2 Disrupt CaM Interaction
Because several LQTS mutations fall within or near the region bordered by the 2 IQ motifs, we next evaluated the effects of a subset of these mutations on the interaction with CaM. We chose to examine S373P in IQ1 (see ref. 29); R518X, which truncates the channel before IQ2 (see ref. 30); and R539W (see ref. 31), which lies just C terminal to IQ2 and thus outside of a predicted CaM interaction domain. Using the strategy detailed in Figure 1, we did not detect any interaction between CaM and the KCNQ1 CT with the S373P mutation (Figure 3A). This mutant KCNQ1 CT was excluded from the soluble fraction of the bacterial lysate, where all of the CaM was found. The expression of the R518X mutant was poor, suggesting that both IQ motifs were necessary for recombinant expression and CaM interaction. Further, CaM1234 did not interact with either construct, suggesting that CaM and CaM1234 bound at the same site. In contrast, the KCNQ1 CT with R539W could be copurified with CaM, similar to the wild-type (WT) KCNQ1 CT. These data suggested that both putative CaM binding sites contributed to a single CaM interaction domain.
CaM binding to the KCNQ1 CT correlated with the resultant functional currents of these LQTS mutants. When expressed in oocytes, KCNQ1 channels with S373P or R518X failed to generate K+ currents different from those recorded from uninjected control oocytes over a range of test potentials from –60 to +60 mV (Figure 3B and 3C); for R518X this was not unexpected, as these channels lack the trafficking domain28 and may also lack key elements that determine assembly (Figure 2F and 2G). R539W channels generated K+ currents (current amplitude was significantly greater than uninjected oocytes for isochronic current amplitude at the end of a 2-second test pulse to +60 mV; n=5 for each, P<0.01), but peak current amplitude was markedly reduced compared with WT channels (19±4%; n=5 for each, P<0.01 at +60 mV) and the V0.5 for activation was 6.9 mV more depolarized (–19.3 mV for WT versus –12.4 mV for R539W). The reduction in peak current, although consistent with the expected loss-of-function phenotype for LQT1 mutations,32 differed from findings in a previous study.31 This may be due, however, to use of different heterologous expression systems. Thus, the functional data suggested that the pathogenesis of at least some LQT1 mutations may be due to their effects on CaM interaction with the KCNQ1 CT.
LQTS Mutations That Disrupt CaM Interaction Are Dominant-Negative
On the basis of the CaM-dependent assembly of KCNQ1 CTs (Figure 2), we hypothesized that defects in channel assembly could explain the pathogenesis of LQTS mutations that disrupt CaM interaction. The specific expectation was that these autosomal dominant mutants would prevent assembly in a dominant-negative manner. We generated a concatemer of a WT and a mutant KCNQ1 subunit, which allowed us to fix the stoichiometry between the WT and mutant channels at 1:1. Although other stoichiometries are possible (ie, 3:1 or 1:3), this method provided a straightforward way to quantitatively assess the effects of including mutant channels within the tetramer and assured that the mutant was expressed in the context of the WT channel, thereby abolishing any concern about differential expression or translation efficiency when WT and mutant channel cRNAs are injected separately. Concatemers of WT KCNQ1 subunits33 generated WT-like currents (Figure 3D). Inclusion of R539W in the concatemer, however, reduced current amplitude, suggesting that these mutant subunits could coassemble with the WT subunits to form functional but impaired heterotetramers (Figure 3E), similar to a previous report.31 When either S373P or R518X were included, the resultant concatemers were not functional, generating currents that were indistinguishable from those recorded from uninjected oocytes (Figure 3E). Neither reversing the order of the WT and mutant subunit within the concatemer (not shown) nor including the accessory subunit KCNE1 (Figure 3F) altered these results. Together, these data suggested that CaM interaction with the KCNQ1 CT correlated with proper channel function and mutations that disrupt this interaction may be associated with LQTS.
KCNQ1 Channels Are Sensitive to Ca2+ and CaM
The interaction of CaM with the KCNQ1 CT and the dominant-negative effect of mutations that abolished CaM interaction prompted us to test next whether associated CaM could provide Ca2+ sensitivity to channel function. A Ca2+-dependent increase of IKs in the heart had been observed19 and attributed to CaM,34 but other studies had suggested different Ca2+-sensitive mediators20 or suggested that KCNQ1 currents were Ca2+ insensitive.23 As a first step, we tested whether changes in intracellular Ca2+ altered channel function by measuring effects on KCNQ1 currents in oocytes after chelation of [Ca2+]i to subphysiological levels by injection of the Ca2+ chelator BAPTA. We recorded currents from the same oocyte 2 minutes after injection of 50 to 100 nL of 100 mmol/L BAPTA (in 10 mmol/L HEPES, pH 7.5), which, in oocytes with a volume of 1 μL, achieved an estimated intracellular BAPTA concentration of 5 to 10 mmol/L. For homomeric KCNQ1 channels, inactivation is readily revealed by a transient increase in the magnitude of the tail current but is not usually obvious during depolarizing pulses.35 Ca2+ chelation, however, unmasked prominent inactivation during the depolarizing pulses (Figure 4A, arrow). Moreover, as seen in Figure 4B, injection of 100 nL BAPTA reduced current amplitude by more than 50% over the entire range of test potentials. In the presence of the subunit KCNE1, which eliminates voltage-dependent inactivation under standard recording conditions,35 chelation of Ca2+ did not produce inactivation during the depolarizing pulses (Figure 4C), although it was even more potent at reducing current amplitude; 100 nL BAPTA reduced current amplitude 85% (Figure 4C). These results were not due to a non-specific effect of BAPTA, as we obtained similar results with EGTA, nor were they due to cell damage during the injection, change in pH, or the result of an increased intracellular osmotic load. Microinjection of 10 mmol/L HEPES with 500 mmol/L sucrose caused no diminution of current (Figure 4A and 4C). Further, these effects were specific to KCNQ1, as we observed no changes in currents in oocytes expressing Cav2.1 Ca2+ channels after an identical BAPTA injection (not shown). Thus, reducing [Ca2+]i acutely to subphysiological levels had pronounced effects on channel inactivation and current amplitude.
We next examined the effects of raising intracellular Ca2+ by injecting 100 nL of 100 μmol/L CaCl2 in 10 mmol/L HEPES. To minimize activation of the endogenous Ca2+-activated Cl– current, we added 0.5 mmol/L niflumic acid to the bath solution. This had no effect on KCNQ1 currents during our standard test protocols (not shown), but was successful in eliminating the contamination of this endogenous current. We observed no significant change in currents after injection of Ca2+ during a repetitive steps to 40 mV for oocytes uninjected with cRNA (Figure 4E). Peak current amplitudes recorded from control oocytes with this protocol ranged from 0.05 to 0.22 μA. In oocytes expressing KCNQ1, however, the same treatment caused a time-dependent increase in peak current that lasted for more than 2 minutes (Figure 4E). This was seen in five different batches of oocytes, and the observed fractional increase was evident when the peak amplitude ranged from 2.6 to 10.7 μA before intracellular injection of CaCl2. These results were consistent with previous reports showing that Ca2+ enhances IKs,19,20,34 and, together with the Ca2+ chelator experiments (Figure 4A through 4D), were the first demonstrations of an effect of Ca2+ on KCNQ1 in a heterologous expression system.
We used two approaches to ascertain whether these Ca2+-regulated effects were mediated by CaM. First, we attempted to replace endogenous CaM with the Ca2+-insensitive CaM1234 by overexpression and to test whether the Ca2+ sensitivity remained. Although coinjection of CaM cRNA increased isochronic current amplitude (52±24% larger at the end of a 2-second test pulse to 60 mV compared with currents from oocytes injected with KCNQ1 only), coinjection of CaM1234 had no effect (Figure 5A) on current amplitude. Coexpression of neither CaM nor CaM1234 altered KCNQ1 current sensitivity to changes in [Ca2+]i. Injection of 50 nL BAPTA caused an 25% reduction of current amplitude whether CaM or CaM1234 cRNA had been coinjected (n=6 for each, P>0.05 for all test potentials between –20 and +50 mV); similarly, coinjection of neither CaM nor CaM1234 cRNA affected the Ca2+-mediated increase in current amplitude over 150 seconds after injection of 100 nL CaCl2 (n=5 to 6, P>0.05 for all time points between 0 and 150 s) or altered inactivation. The absence of an apparent effect with CaM1234 coinjection could not be attributed to ineffectual CaM1234 expression because in control experiments, this same cRNA effectively blocked Ca2+/CaM-mediated Ca2+-dependent inactivation of Cav1.2 Ca2+ channels (not shown). Instead, we expected that the ineffectiveness of CaM1234 could be explained by a failure of CaM1234 to compete with endogenous CaM, as suggested by the apparent higher affinity of CaM for the KCNQ1 CT compared with CaM1234 (Figure 2E). We therefore used a concatemer strategy, linking CaM1234 to the KCNQ1 N terminus to increase its local concentration in the vicinity of KCNQ1 and provide a more effective competitor for endogenous CaM. This also restricted CaM1234 to the channel and thus avoided any pleiotropic effects of CaM1234 overexpression on other CaM-signaling pathways. CaM1234–KCNQ1 concatemers revealed prominent inactivation during the depolarizing pulse (Figure 5B, arrow), similar to that observed with Ca2+ chelation (Figure 4B). CaM–KCNQ1 concatemers, in contrast, produced currents similar to those from WT KCNQ1 and revealed inactivation only in the transient increase in tail currents (Figure 5B). A quantitative assessment of inactivation was made by comparing the ratio of the peak current during the first 50 ms to the current at the end of the 2-second depolarizing pulse (Figure 5C). For currents from KCNQ1 channels or CaM-KCNQ1 channels, current amplitude continued to rise after the first 50 ms, so that the ratio was significantly less than unity. For currents from CaM1234–KCNQ1 channels or KCNQ1 channels after BAPTA injection (Figure 4B), prominent inactivation during the depolarizing pulse increased the ratio significantly.
Discussion
Formation of ion channels requires a complex series of coordinated events, beginning with protein synthesis and ending with insertion of functional channels into the appropriate target membrane. For channels that function as multimeric complexes, subunit assembly with the appropriate stoichiometry and types of auxiliary subunits occurs early in this process in the endoplasmic reticulum.36 CaM has been recognized as a dissociable subunit for several ion channels and as a constitutive subunit for others,37 and thereby confers on mature channels that have reached their target membrane Ca2+ dependence to a variety of functions, such as channel activation9 or inactivation.10,12 In those cases in which CaM is a constitutive subunit, its presence during channel formation affords the opportunity to confer regulation additionally to channel biosynthesis, as has been previously shown for trafficking of SK K+ channels.38,39
The data presented here showing that CaM participates in assembly of KCNQ1 homotetramers suggest that CaM regulation of channel formation may be widespread and therefore add to the diversity of roles CaM, as an auxiliary subunit, plays in ion channel regulation. The implications for disease are extensive. At a general level, these findings suggest new approaches to understanding pathophysiology in the many disease states in which alterations in levels of Ca2+ and CaM have been found and effects on channel function have been described, such as epilepsy and heart failure40–44 In the particular case of CaM interaction with KCNQ1, we also provide a new example of an ion channel defect that leads to LQTS: mutations that disrupt CaM interaction block proper channel assembly in a dominant-negative fashion. A role for CaM in channel assembly is consistent with studies that showed that determinants proposed to govern KCNQ2/KCNQ3 subunit interaction fall within the homologous CaM binding domain45 and therefore likely also explains the etiology of epileptogenic mutations in KCNQ2 that fall within this region.46–49 Indeed, two of these epileptogenic mutants46,47 and several artificial KCNQ2 mutants22 have been tested functionally and, similar to what we observed for the CaM binding mutants in KCNQ1 (Figure 3B and 3C), these mutant KCNQ2 channels did not yield K+ currents.
We also demonstrated that a stoichiometry of one CaM per KCNQ1 CT is sufficient for interaction, even though mutations in both IQ motifs result in loss of CaM binding. The possibility that both IQ motifs contribute to a complex CaM binding pocket is entirely consistent with structural data showing non-canonical interactions between CaM and multiple domains of target proteins.50 The ability of CaM1234, a surrogate for Ca2+-free apocalmodulin, to bind to the KCNQ1 CT is further indication that the interaction between CaM and KCNQ1 is non-canonical.
Our data also provide new evidence that Ca2+ regulates KCNQ1 currents and are the first data to show Ca2+ sensitivity of KCNQ1 in a heterologous expression system. The most striking finding was that chelation of [Ca2+]i below physiological levels or the incorporation of a Ca2+-insensitive CaM induced prominent inactivation during the depolarizing pulses and reduced current amplitude. These two phenomena may not be directly coupled, as Ca2+ chelation reduced current amplitude from channels expressing KCNQ1+KCNE1 but did not induce prominent inactivation. This alteration in inactivation may have important physiological effects outside the heart, where KCNQ1 is not invariably associated with KCNE1. Another finding was that increases in [Ca2+]i above basal levels enhance KCNQ1 current. The mechanism for this is not yet clear, but it could involve partial relief of inactivation. Thus, data presented here offer new insights into mechanisms of cardiac arrhythmias and epilepsy, two major disorders that result from mutations in the KCNQ family of K+ channels, opening an important window onto the elusive triggers that cause the rare and episodic arrhythmias or seizures in patients carrying mutations in these channels.
Acknowledgments
This work was supported by grants from the National Institutes of Health and American Heart Association to G.S.P. and the Irma T. Hirschl Monique Weill-Caulier Trust. G.S.P. is the Esther Aboodi Assistant Professor of Medicine. S.G. was supported by a training grant from the National Institutes of Health. We thank Ming Zhou for advice on DSG cross-linking and Lillian Seu for help with recordings.
Footnotes
Original received October 29, 2005; revision received February 9, 2006; accepted February 23, 2006.
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the Departments of Pharmacology (S.G., D.A.N., G.S.P.) and Medicine, Division of Cardiology (G.S.P.), College of Physicians and Surgeons of Columbia University, New York, NY.
Abstract
Calmodulin (CaM) has been recognized as an obligate subunit for many ion channels in which its function has not been clearly established. Because channel subunits associate early during channel biosynthesis, CaM may provide a mechanism for Ca2+-dependent regulation of channel formation. Here we show that CaM is a constitutive component of KCNQ1 K+ channels, the most commonly mutated long-QT syndrome (LQTS) locus. CaM not only acts as a regulator of channel gating, relieving inactivation in a Ca2+-dependent manner, but it also contributes to control of channel assembly. Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus. This CaM-regulated process is essential: LQTS mutants that disrupt CaM interaction prevent functional assembly of channels in a dominant-negative manner. These findings offer a new mechanism for LQTS defects and provide a basis for understanding novel ways that intracellular Ca2+ and CaM regulate ion channels.
Key Words: KCNQ1 KvLQT1 IKs calmodulin long-QT syndrome
Introduction
The long-QT syndrome (LQTS) is a collection of inherited and acquired arrhythmogenic diseases characterized on ECG by a prolonged QT interval. This ECG manifestation reflects an abnormally prolonged ventricular action potential, which can be the substrate for life-threatening arrhythmias that lead to syncope or sudden cardiac death. LQTS is a "channelopathy;"all except one LQTS loci reside in genes encoding ion channels that regulate the cardiac action potential, and characterization of LQTS mutations in specific channels reveals defects that promote excessive depolarization or weakened repolarization.1–3 Nevertheless, development of arrhythmias is rare, and the specific triggers that induce them are not well understood.
Intracellular Ca2+ is one factor with a large potential impact that has only recently been explored.3–7 As Ca2+ is the fundamental second messenger of electrical activity in excitable cells, it is not surprising that many ion channels are subject to feedback modulation by resultant Ca2+ fluxes, a process often mediated by the ubiquitous Ca2+-binding protein calmodulin (CaM).8–13 The recent identification of two possible CaM interaction sites within the C-terminus of KCNQ1,14 the most commonly mutated locus (LQT1) in congenital LQTS,15 offered a window into how Ca2+ may contribute to LQTS. As the pore-forming subunit of the catecholamine-sensitive cardiac IKs current that determines action potential duration on a beat-to-beat basis,16,17 KCNQ1 is well positioned to integrate changes in intracellular Ca2+ into an alteration in action potential duration, consistent with previous reports showing that IKs is a Ca2+-responsive current.18–20 Moreover, the neuronal homologs KCNQ2 and KCNQ3 form heteromultimers that are responsible for the Ca2+-sensitive M current21 in the brain and have also been shown to bind CaM,14,22 suggesting that Ca2+/CaM could be a general regulator for the KCNQ K+ channel family. Nevertheless, attempts to define whether and how Ca2+/CaM regulates any of the KCNQ homologs have produced conflicting results.22–24 Moreover, KCNQ1 stood apart in these initial studies because Ca2+/CaM did not appear to regulate KCNQ1 or IKs currents in a heterologous system and because CaM was not found to interact with KCNQ1. Here, we report that CaM binds to the KCNQ1 C-terminus, where it contributes to channel assembly. On mature channels, CaM appears to relieve channel inactivation in the presence of basal levels of intracellular Ca2+. Further relief of inactivation may augment KCNQ1 current when Ca2+ is elevated.
Materials and Methods
Additional Materials and Methods are available in the online-only data supplement at http://circres.ahajournals.org.
Molecular Biology
Bacterial and oocyte expression constructs were generated with standard approaches. Cloning of the KCNQ1 concatemer yielded Gly-Ser dipeptide between the 2 KCNQ1 repeats.
Electrophysiology
Two electrode voltage-clamp recordings from Xenopus oocytes were preformed as previously described25 with the use of ND-96 bath solution. Intracellular Ca2+ manipulations were performed by micro-injection of 50 to 100 nL of 100 mmol/L BAPTA, 100 mmol/L EGTA, 100 μmol/L CaCl2, or 500 mmol/L sucrose (in 10 mmol/L HEPES, pH 7.4).
Protein Expression and Purification
The KCNQ1 CT constructs, with and without CaM or CaM1234, were isolated from BL-21 (DE3) cells and purified with Talon metal affinity resin (Clontech). Gel filtration was performed over a Superdex 200 HR 10/30 column on an AKTA FPLC (Amersham Biosciences) in 500 mmol/L NaCl, 20 mmol/L Tris, pH 7.5, and 10 μmol/L CaCl2.
Chemical Cross-Linking
Disuccinimidyl glutarate 2.5 mmol/L was added for 5 minutes at room temperature to the purified KCNQ1 CT/CaM complexes before the addition of SDS-PAGE sample buffer.
Statistical Analysis
Data are presented as mean±SEM. Significant differences were assessed using Student t test at P<0.05.
Results
CaM Interacts Directly With the KCNQ1 C-Terminus
To directly assess whether CaM interacted with the KCNQ1 CT, we used paradigms successful in defining CaM interaction with other voltage-gated ion channels.6,26 We first attempted to generate recombinant protein under detergent-free conditions for quantitative binding assays with a 6xHis-tagged KCNQ1 construct of the entire CT (aa 354–676; Figure 1A), but despite abundant protein expression, none was soluble (Figure 1B). This result was similar to that observed for the CaM-binding regions in the CTs of Cav1.2, Nav1.2, and Nav1.5 channels, so we coexpressed CaM with the KCNQ1 CT and found that the KCNQ1 CT became soluble (Figure 1B). Not only did CaM promote KCNQ1 CT solubility, but it was also a KCNQ1 CT binding partner, as indicated by CaM copurification with the 6xHis-tagged KCNQ1 CT by metal affinity chromatography (MAC) (Figure 1B). This interaction was specific, as CaM was not purified in the absence of KCNQ1 CT coexpression (not shown). Further, this interaction could be Ca2+-independent, as CaM1234, a CaM mutant deficient in Ca2+ binding,9 also copurified when coexpressed with the KCNQ1 CT (Figure 1B; see below). The interaction also was Mg2+-independent, as no Mg2+ was added to the buffers. Although consistent with previous findings that CaM interacts with the CTs of other KCNQ family members,14,22,24 this was the first direct demonstration that CaM interacted with KCNQ1.
The Stoichiometry Between CaM and the KCNQ1 CT is 1:1
Although KCNQ CTs contain two potential CaM binding sites (IQ1 and IQ2 in Figure 1A),14 the stoichiometry between CaM and any KCNQ channel has not been determined. As a first estimate, we evaluated the purified KCNQ1 CT/CaM complex on size exclusion chromatography. The complex ran as a single species with an apparent Mw significantly greater than the sum of the 6xHis-tagged KCNQ1 CT and CaM (Figure 2A). The elution profile for the complex was unchanged whether saturating Ca2+ (1 mmol/L) or EGTA (5 mmol/L) was added to the column buffer, further supporting the hypothesis that the interaction between CaM and the KCNQ1 CT did not require Ca2+ (but see below). These data, however, did not yield an accurate assessment of the complex’s Mw or the stoichiometry between CaM and KCNQ1 CT. We therefore performed chemical cross-linking with disuccinimidyl glutarate (DSG), a homobifunctional, non-cleavable reagent reactive toward free amino groups, and subsequent polyacrylamide gel electrophoresis. After 5 minutes’ exposure to DSG at 25°C, none of the 36 kDa KCNQ1 CT input material remained; instead, a single predominant band migrating at 240 kDa was visible after Coomassie staining (Figure 2B, left). CaM was not visible in the input material (lane 1), having migrated off the 7% polyacrylamide gel (arrow depicts the gel’s dye-front), but it could be readily detected in the higher Mw cross-linked material by immunoblot (Figure 2B, right). Similar results were obtained with the KCNQ1 CT/CaM1234 complex (Figure 2B, right). The apparent Mw of the complex, which included both CaM and the KCNQ1 C-terminus, was less than 250 kDa on SDS-PAGE and was therefore consistent with a 4:4 (KCNQ1 CT:CaM) heteromultimer (estimated Mw of 212 kDa), rather than a 4:8 heteromultimer (estimated Mw of 280 kDa). This was consistent with one CaM molecule bound for each putative CaM binding site.
To establish whether one CaM molecule per KCNQ1 CT was sufficient, we fixed the stoichiometry at 1:1 by expressing a construct in which CaM or CaM1234 was joined by a 12x glycine (G12) linker at the KCNQ1 construct’s CT (CT-G12-CaM or CT-G12-CaM1234). These constructs were soluble without the coexpression of unlinked CaM (Figure 2C), and DSG cross-linking of the purified material yielded a product that migrated at 4x the Mw of the untreated material (Figure 2D). The concatemers alo allowed us to determine relative affinities of CaM and CaM1234 for the KCNQ1 CT. When coexpressed with the CT-G12-CaM construct, unlinked CaM, but not unlinked CaM1234, co-purified on MAC (Figure 2E, arrow). This suggested that unlinked CaM but not CaM1234, could complete with linked CaM for association with the KCNQ1 CT. Thus, although CaM could bind to the KCNQ1 CT in the absence of Ca2+, Ca2+/CaM had a higher relative affinity.
We used the in vitro formation of the KCNQ1 CT tetramer to test definitively whether amino acids 589 to 620 were necessary for assembly, as this region had been previously identified as the KCNQ1 assembly domain,27 but a recent study suggested instead that it functioned as a trafficking signal.28 We generated a KCNQ1 CT truncated after amino acid 555 (555, Mw 24 kDa) to exclude 589 to 620. As with the full length KCNQ1 CT, the 555 required CaM coexpression to generate soluble material suitable for MAC purification (not shown). Chemical cross-linking with DSG generated products of 45 kDa, consistent with the inclusion of one 555 KCNQ1 CT and one CaM molecule (Figure 2F, single arrowhead); 100 kDa, consistent with a dimer of the 555 KCNQ1 CT/CaM complex (Figure 2F, double arrowhead); and 200 kDa, consistent with a tetramer of complexes (Figure 2F, asterisk). These results suggested that the KCNQ1 CT/CaM complexes were able to assemble independent of amino acids 589 to 620.
LQTS Mutations in Either IQ1 or IQ2 Disrupt CaM Interaction
Because several LQTS mutations fall within or near the region bordered by the 2 IQ motifs, we next evaluated the effects of a subset of these mutations on the interaction with CaM. We chose to examine S373P in IQ1 (see ref. 29); R518X, which truncates the channel before IQ2 (see ref. 30); and R539W (see ref. 31), which lies just C terminal to IQ2 and thus outside of a predicted CaM interaction domain. Using the strategy detailed in Figure 1, we did not detect any interaction between CaM and the KCNQ1 CT with the S373P mutation (Figure 3A). This mutant KCNQ1 CT was excluded from the soluble fraction of the bacterial lysate, where all of the CaM was found. The expression of the R518X mutant was poor, suggesting that both IQ motifs were necessary for recombinant expression and CaM interaction. Further, CaM1234 did not interact with either construct, suggesting that CaM and CaM1234 bound at the same site. In contrast, the KCNQ1 CT with R539W could be copurified with CaM, similar to the wild-type (WT) KCNQ1 CT. These data suggested that both putative CaM binding sites contributed to a single CaM interaction domain.
CaM binding to the KCNQ1 CT correlated with the resultant functional currents of these LQTS mutants. When expressed in oocytes, KCNQ1 channels with S373P or R518X failed to generate K+ currents different from those recorded from uninjected control oocytes over a range of test potentials from –60 to +60 mV (Figure 3B and 3C); for R518X this was not unexpected, as these channels lack the trafficking domain28 and may also lack key elements that determine assembly (Figure 2F and 2G). R539W channels generated K+ currents (current amplitude was significantly greater than uninjected oocytes for isochronic current amplitude at the end of a 2-second test pulse to +60 mV; n=5 for each, P<0.01), but peak current amplitude was markedly reduced compared with WT channels (19±4%; n=5 for each, P<0.01 at +60 mV) and the V0.5 for activation was 6.9 mV more depolarized (–19.3 mV for WT versus –12.4 mV for R539W). The reduction in peak current, although consistent with the expected loss-of-function phenotype for LQT1 mutations,32 differed from findings in a previous study.31 This may be due, however, to use of different heterologous expression systems. Thus, the functional data suggested that the pathogenesis of at least some LQT1 mutations may be due to their effects on CaM interaction with the KCNQ1 CT.
LQTS Mutations That Disrupt CaM Interaction Are Dominant-Negative
On the basis of the CaM-dependent assembly of KCNQ1 CTs (Figure 2), we hypothesized that defects in channel assembly could explain the pathogenesis of LQTS mutations that disrupt CaM interaction. The specific expectation was that these autosomal dominant mutants would prevent assembly in a dominant-negative manner. We generated a concatemer of a WT and a mutant KCNQ1 subunit, which allowed us to fix the stoichiometry between the WT and mutant channels at 1:1. Although other stoichiometries are possible (ie, 3:1 or 1:3), this method provided a straightforward way to quantitatively assess the effects of including mutant channels within the tetramer and assured that the mutant was expressed in the context of the WT channel, thereby abolishing any concern about differential expression or translation efficiency when WT and mutant channel cRNAs are injected separately. Concatemers of WT KCNQ1 subunits33 generated WT-like currents (Figure 3D). Inclusion of R539W in the concatemer, however, reduced current amplitude, suggesting that these mutant subunits could coassemble with the WT subunits to form functional but impaired heterotetramers (Figure 3E), similar to a previous report.31 When either S373P or R518X were included, the resultant concatemers were not functional, generating currents that were indistinguishable from those recorded from uninjected oocytes (Figure 3E). Neither reversing the order of the WT and mutant subunit within the concatemer (not shown) nor including the accessory subunit KCNE1 (Figure 3F) altered these results. Together, these data suggested that CaM interaction with the KCNQ1 CT correlated with proper channel function and mutations that disrupt this interaction may be associated with LQTS.
KCNQ1 Channels Are Sensitive to Ca2+ and CaM
The interaction of CaM with the KCNQ1 CT and the dominant-negative effect of mutations that abolished CaM interaction prompted us to test next whether associated CaM could provide Ca2+ sensitivity to channel function. A Ca2+-dependent increase of IKs in the heart had been observed19 and attributed to CaM,34 but other studies had suggested different Ca2+-sensitive mediators20 or suggested that KCNQ1 currents were Ca2+ insensitive.23 As a first step, we tested whether changes in intracellular Ca2+ altered channel function by measuring effects on KCNQ1 currents in oocytes after chelation of [Ca2+]i to subphysiological levels by injection of the Ca2+ chelator BAPTA. We recorded currents from the same oocyte 2 minutes after injection of 50 to 100 nL of 100 mmol/L BAPTA (in 10 mmol/L HEPES, pH 7.5), which, in oocytes with a volume of 1 μL, achieved an estimated intracellular BAPTA concentration of 5 to 10 mmol/L. For homomeric KCNQ1 channels, inactivation is readily revealed by a transient increase in the magnitude of the tail current but is not usually obvious during depolarizing pulses.35 Ca2+ chelation, however, unmasked prominent inactivation during the depolarizing pulses (Figure 4A, arrow). Moreover, as seen in Figure 4B, injection of 100 nL BAPTA reduced current amplitude by more than 50% over the entire range of test potentials. In the presence of the subunit KCNE1, which eliminates voltage-dependent inactivation under standard recording conditions,35 chelation of Ca2+ did not produce inactivation during the depolarizing pulses (Figure 4C), although it was even more potent at reducing current amplitude; 100 nL BAPTA reduced current amplitude 85% (Figure 4C). These results were not due to a non-specific effect of BAPTA, as we obtained similar results with EGTA, nor were they due to cell damage during the injection, change in pH, or the result of an increased intracellular osmotic load. Microinjection of 10 mmol/L HEPES with 500 mmol/L sucrose caused no diminution of current (Figure 4A and 4C). Further, these effects were specific to KCNQ1, as we observed no changes in currents in oocytes expressing Cav2.1 Ca2+ channels after an identical BAPTA injection (not shown). Thus, reducing [Ca2+]i acutely to subphysiological levels had pronounced effects on channel inactivation and current amplitude.
We next examined the effects of raising intracellular Ca2+ by injecting 100 nL of 100 μmol/L CaCl2 in 10 mmol/L HEPES. To minimize activation of the endogenous Ca2+-activated Cl– current, we added 0.5 mmol/L niflumic acid to the bath solution. This had no effect on KCNQ1 currents during our standard test protocols (not shown), but was successful in eliminating the contamination of this endogenous current. We observed no significant change in currents after injection of Ca2+ during a repetitive steps to 40 mV for oocytes uninjected with cRNA (Figure 4E). Peak current amplitudes recorded from control oocytes with this protocol ranged from 0.05 to 0.22 μA. In oocytes expressing KCNQ1, however, the same treatment caused a time-dependent increase in peak current that lasted for more than 2 minutes (Figure 4E). This was seen in five different batches of oocytes, and the observed fractional increase was evident when the peak amplitude ranged from 2.6 to 10.7 μA before intracellular injection of CaCl2. These results were consistent with previous reports showing that Ca2+ enhances IKs,19,20,34 and, together with the Ca2+ chelator experiments (Figure 4A through 4D), were the first demonstrations of an effect of Ca2+ on KCNQ1 in a heterologous expression system.
We used two approaches to ascertain whether these Ca2+-regulated effects were mediated by CaM. First, we attempted to replace endogenous CaM with the Ca2+-insensitive CaM1234 by overexpression and to test whether the Ca2+ sensitivity remained. Although coinjection of CaM cRNA increased isochronic current amplitude (52±24% larger at the end of a 2-second test pulse to 60 mV compared with currents from oocytes injected with KCNQ1 only), coinjection of CaM1234 had no effect (Figure 5A) on current amplitude. Coexpression of neither CaM nor CaM1234 altered KCNQ1 current sensitivity to changes in [Ca2+]i. Injection of 50 nL BAPTA caused an 25% reduction of current amplitude whether CaM or CaM1234 cRNA had been coinjected (n=6 for each, P>0.05 for all test potentials between –20 and +50 mV); similarly, coinjection of neither CaM nor CaM1234 cRNA affected the Ca2+-mediated increase in current amplitude over 150 seconds after injection of 100 nL CaCl2 (n=5 to 6, P>0.05 for all time points between 0 and 150 s) or altered inactivation. The absence of an apparent effect with CaM1234 coinjection could not be attributed to ineffectual CaM1234 expression because in control experiments, this same cRNA effectively blocked Ca2+/CaM-mediated Ca2+-dependent inactivation of Cav1.2 Ca2+ channels (not shown). Instead, we expected that the ineffectiveness of CaM1234 could be explained by a failure of CaM1234 to compete with endogenous CaM, as suggested by the apparent higher affinity of CaM for the KCNQ1 CT compared with CaM1234 (Figure 2E). We therefore used a concatemer strategy, linking CaM1234 to the KCNQ1 N terminus to increase its local concentration in the vicinity of KCNQ1 and provide a more effective competitor for endogenous CaM. This also restricted CaM1234 to the channel and thus avoided any pleiotropic effects of CaM1234 overexpression on other CaM-signaling pathways. CaM1234–KCNQ1 concatemers revealed prominent inactivation during the depolarizing pulse (Figure 5B, arrow), similar to that observed with Ca2+ chelation (Figure 4B). CaM–KCNQ1 concatemers, in contrast, produced currents similar to those from WT KCNQ1 and revealed inactivation only in the transient increase in tail currents (Figure 5B). A quantitative assessment of inactivation was made by comparing the ratio of the peak current during the first 50 ms to the current at the end of the 2-second depolarizing pulse (Figure 5C). For currents from KCNQ1 channels or CaM-KCNQ1 channels, current amplitude continued to rise after the first 50 ms, so that the ratio was significantly less than unity. For currents from CaM1234–KCNQ1 channels or KCNQ1 channels after BAPTA injection (Figure 4B), prominent inactivation during the depolarizing pulse increased the ratio significantly.
Discussion
Formation of ion channels requires a complex series of coordinated events, beginning with protein synthesis and ending with insertion of functional channels into the appropriate target membrane. For channels that function as multimeric complexes, subunit assembly with the appropriate stoichiometry and types of auxiliary subunits occurs early in this process in the endoplasmic reticulum.36 CaM has been recognized as a dissociable subunit for several ion channels and as a constitutive subunit for others,37 and thereby confers on mature channels that have reached their target membrane Ca2+ dependence to a variety of functions, such as channel activation9 or inactivation.10,12 In those cases in which CaM is a constitutive subunit, its presence during channel formation affords the opportunity to confer regulation additionally to channel biosynthesis, as has been previously shown for trafficking of SK K+ channels.38,39
The data presented here showing that CaM participates in assembly of KCNQ1 homotetramers suggest that CaM regulation of channel formation may be widespread and therefore add to the diversity of roles CaM, as an auxiliary subunit, plays in ion channel regulation. The implications for disease are extensive. At a general level, these findings suggest new approaches to understanding pathophysiology in the many disease states in which alterations in levels of Ca2+ and CaM have been found and effects on channel function have been described, such as epilepsy and heart failure40–44 In the particular case of CaM interaction with KCNQ1, we also provide a new example of an ion channel defect that leads to LQTS: mutations that disrupt CaM interaction block proper channel assembly in a dominant-negative fashion. A role for CaM in channel assembly is consistent with studies that showed that determinants proposed to govern KCNQ2/KCNQ3 subunit interaction fall within the homologous CaM binding domain45 and therefore likely also explains the etiology of epileptogenic mutations in KCNQ2 that fall within this region.46–49 Indeed, two of these epileptogenic mutants46,47 and several artificial KCNQ2 mutants22 have been tested functionally and, similar to what we observed for the CaM binding mutants in KCNQ1 (Figure 3B and 3C), these mutant KCNQ2 channels did not yield K+ currents.
We also demonstrated that a stoichiometry of one CaM per KCNQ1 CT is sufficient for interaction, even though mutations in both IQ motifs result in loss of CaM binding. The possibility that both IQ motifs contribute to a complex CaM binding pocket is entirely consistent with structural data showing non-canonical interactions between CaM and multiple domains of target proteins.50 The ability of CaM1234, a surrogate for Ca2+-free apocalmodulin, to bind to the KCNQ1 CT is further indication that the interaction between CaM and KCNQ1 is non-canonical.
Our data also provide new evidence that Ca2+ regulates KCNQ1 currents and are the first data to show Ca2+ sensitivity of KCNQ1 in a heterologous expression system. The most striking finding was that chelation of [Ca2+]i below physiological levels or the incorporation of a Ca2+-insensitive CaM induced prominent inactivation during the depolarizing pulses and reduced current amplitude. These two phenomena may not be directly coupled, as Ca2+ chelation reduced current amplitude from channels expressing KCNQ1+KCNE1 but did not induce prominent inactivation. This alteration in inactivation may have important physiological effects outside the heart, where KCNQ1 is not invariably associated with KCNE1. Another finding was that increases in [Ca2+]i above basal levels enhance KCNQ1 current. The mechanism for this is not yet clear, but it could involve partial relief of inactivation. Thus, data presented here offer new insights into mechanisms of cardiac arrhythmias and epilepsy, two major disorders that result from mutations in the KCNQ family of K+ channels, opening an important window onto the elusive triggers that cause the rare and episodic arrhythmias or seizures in patients carrying mutations in these channels.
Acknowledgments
This work was supported by grants from the National Institutes of Health and American Heart Association to G.S.P. and the Irma T. Hirschl Monique Weill-Caulier Trust. G.S.P. is the Esther Aboodi Assistant Professor of Medicine. S.G. was supported by a training grant from the National Institutes of Health. We thank Ming Zhou for advice on DSG cross-linking and Lillian Seu for help with recordings.
Footnotes
Original received October 29, 2005; revision received February 9, 2006; accepted February 23, 2006.
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