Delayed rectifier K+ currents and cardiac repolarization

Journal of Molecular and Cellular Cardiology 48 (2010) 37–44

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c

Review article

Delayed rectifier K+ currents and cardiac repolarization Flavien Charpentier a,b,c,d, Jean Mérot a,b,c, Gildas Loussouarn a,b,c, Isabelle Baró a,b,c,⁎ a

INSERM, UMR915, l'institut du thorax, Nantes, F-44000, France CNRS, ERL3147, Cardiopathies et mort subite, Nantes, F-44000, France c Université de Nantes, Nantes, F-44000, France d CHU Nantes, l'institut du thorax, Nantes, F-44000, France b

a r t i c l e

i n f o

Article history: Received 25 May 2009 Received in revised form 16 July 2009 Accepted 6 August 2009 Available online 14 August 2009 Keywords: K+ currents Heart Repolarization

a b s t r a c t The two components of the cardiac delayed rectifier current have been the subject of numerous studies since firstly described. This current controls the action potential duration and is highly regulated. After identification of the channel subunits underlying IKs, KCNQ1 associated with KCNE1, and IKr, HERG, their involvement in human cardiac channelopathies have provided various models allowing the description of the molecular mechanisms of the KCNQ1 and HERG channels trafficking, activity and regulation. More recently, studies have been focusing on the unveiling of different partners of the pore-forming proteins that contribute to their maturation, trafficking, activity and/or degradation, on one side, and on their respective expression in the heterogeneous cardiac tissue, on the other side. The aim of this review is to report and discuss the major works on IKs and IKr and the most recent ones that help to understand the precise function of these currents in the heart. © 2009 Elsevier Inc. All rights reserved.

Contents 1.

The slow component, IKs . . . . . . . . . . . . . . . . . . . 1.1. Molecular identification of the IKs-related channel . . . . 1.2. IKs subunits, from pathologies to structure . . . . . . . 1.2.1. Loss of function mutations and tetramerization . 1.2.2. RW mutations and trafficking . . . . . . . . . 1.2.3. KCNQ1 gain-of-function mutations and KCNE1 . 1.2.4. KCNE subunits: distribution and function. . . . 1.2.5. KCNQ1 auto-regulation of channel trafficking . . 1.3. IKs regulation . . . . . . . . . . . . . . . . . . . . . 1.3.1. AKAPs and PKA-dependent regulation of IKs . . 1.3.2. Calmodulin-dependent regulation . . . . . . . 1.3.3. Nedd4-2 and KCNQ1 degradation . . . . . . . 1.3.4. PIP2 regulation of KCNQ1 . . . . . . . . . . . 2. The fast component IKr . . . . . . . . . . . . . . . . . . . . 2.1. Molecular identification of IKr-related channel, HERG . . 2.2. Uniqueness of IKr among voltage-dependent K+ currents 2.3. SQT1 mutations and inactivation . . . . . . . . . . . . 2.4. LQT2 mutations and trafficking . . . . . . . . . . . . . 2.5. HERG1a/1b and trafficking . . . . . . . . . . . . . . . 2.6. HERG partners and IKr regulation . . . . . . . . . . . . 2.6.1. KCR1 and HERG pharmacological response . . . 2.6.2. ARHGAP6, PIP2 and HERG . . . . . . . . . . . 3. KCNQ1 and HERG are partners . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. INSERM UMR915, CNRS ERL3147, l'institut du thorax, IRT – UN, 8, quai Moncousu, BP 70721, 44007 Nantes cedex 1, France. Tel. +33 228 08 01 50; fax: +33 228 08 0130. E-mail address: [email protected] (I. Baró). 0022-2828/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2009.08.005

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The delayed rectifier K+ current, IK, first described by Noble and Tsien [1,2], is responsible for the late repolarization phase of the action potential (AP) and regulates AP duration (APD) in many species. In these first publications, the two components of this voltage-dependent

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current were described after a kinetic analysis. A work published in 1990 by Sanguinetti and Jurkievicz in guinea pig ventricular cardiomyocytes confirmed IK heterogeneity using pharmacological dissection [3]. Indeed, the two components differentiate by their kinetics and sensitivity to the benzenesulfonamide compound, E-4031. The druginsensitive component, IKs, shows a very slow activation whereas the E4131-sensitive component, IKr, activates more rapidly and exhibits a remarkable prominent inward rectification. IKs and IKr respective contribution to the AP repolarization varies among species [4], and the role of IKs in the human ventricular AP has been debated. A very elegant demonstration of the respective contribution of IKs and IKr to the normal ventricular human AP in the absence of tonic sympathetic stimulation was provided by Jost et al. [5] in 2005. Their work shows that pharmacological IKs block plays little role in increasing normal human ventricular APD. However, when repolarization reserve is attenuated, IKs plays an increasingly important role in limiting AP prolongation. APD is very tightly tuned via IKs and IKr regulation. IKs contribution is enhanced at high stimulation rates due to the incomplete slow deactivation of the current [6]. Catecholamines shorten APD by a βadrenoceptor-mediated increase in the magnitude of IKs through the PKA pathway [7,8]. IKs is also modulated in the heart by α-adrenergic receptors through the PKC pathway [9,10]. On the other hand, IKr response to adrenergic signaling is more complex [8,11]. In this review, we will present current knowledge on the proteins generating these currents and we will focus on newly identified proteins associating with the channels and contributing to their regulation. 1. The slow component, IKs 1.1. Molecular identification of the IKs-related channel The molecular identification of the K+-channel-generating IKs was not straightforward. First it was attributed to a small single transmembrane domain protein, firstly called minK or IsK (now called KCNE1), that was able to generate a large voltage-dependent K+ current reminiscent of IKs when re-expressed in Xenopus oocytes [12,13]. These results were highly debated because the size and structure of this protein are such that it was unlikely that KCNE1 alone formed functional channels and because KCNE1 expression failed to generate any current in most cellular expression systems. KCNE1 actual function was revealed years later by co-expression with the newly cloned K+ channel KvLQT1 [14] (now called KCNQ1 or Kv7.1) [15,16]. KCNQ1 gene was detected with a positional cloning approach, linked to the inherited long QT (LQT) syndrome form 1. LQT syndrome is a cardiac disorder characterized by abnormally prolonged ventricular repolarization that results in episodic ventricular tachyarrhythmias sometimes leading to ventricular fibrillation and sudden death in otherwise healthy persons [17]. Hydrophobicity analysis of KCNQ1 gene product predicted a classic voltage-dependent K+ channel (Kv) topology with six transmembrane segments. As for other Kv-type channels, the pore unit is constituted by tetramerization of KCNQ1. In mammalian cells, expression of KCNQ1 alone gave rise to a fast activating K+ current and KCNE1 co-expression was required for the current to recapitulate cardiac IKs. KCNE1 considerably increased the current amplitude, delayed its activation and shifted voltage dependence of activation. It is now well established that KCNE1 is a βsubunit of the channel complex underlying the cardiac IKs that regulates the KCNQ1 α-subunit trafficking [18] and behavior [15,16]. Mutations of KCNE1 gene are also associated with LQT syndrome (LQT5) [19,20]. 1.2. IKs subunits, from pathologies to structure KCNQ1 and KCNE1 cloning and heterologous expression allowed numerous investigations to understand IKs biophysics (for review, see

[21]). Valuable information was also provided by studies of naturally occurring mutations related to human familial diseases that allowed better understanding of LQT genotype-phenotype and structurefunction relationship of the cardiac KCNQ1–KCNE1 complex. 1.2.1. Loss of function mutations and tetramerization LQT-related mutations are characterized by IKs loss of function resulting from trafficking defect to dysfunction or dysregulation at the membrane (for recent reviews, see [22,23]). Two different forms of LQT1 have been reported: the autosomal dominant Romano–Ward syndrome (RW) and the autosomal recessive Jervell and Lange– Nielsen syndrome (JLN), which associates bilateral deafness with the cardiac disease [24]. Functional studies of JLN- and RW-associated KCNQ1 mutations highlighted the role of a C-terminus domain of KCNQ1 in the tetramer assembly. Indeed, it is now clear that JLNmutated KCNQ1 subunits are devoid of significant, if any, dominantnegative effect on the wild-type (WT) subunits unlike RW ones [25]. RW KCNQ1 impairs WT KCNQ1 channel activity, whereas JLN KCNQ1 co-expression with WT channel has no major effect on the resulting current. A large number of JLN mutations results in truncation of the C-terminus or missense sequences located in the C-terminal assembly A-domain and prevents the formation of WT–mutant heterotetramers [26–28]. However, JLN missense mutations in this domain may act through a different pathophysiological mechanism. Minor et al. clearly showed that the A-domain bearing JLN mutations (T587M and G589D) can tetramerize. Even if they did not look at WT–mutant (but only pure WT or pure mutant) tetramerization of the A-domain, their work suggests that full-length mutant channel can associate to WT channel but that WT–mutant heterotetramers cannot reach the plasma membrane due to the quality control of the trafficking machinery. One explanation may be the lack of association with the AKAP yotiao ([29] and cf. below). This leads to the important idea that JLN is not always correlated with tetramerization deficiency of the mutant channel. 1.2.2. RW mutations and trafficking Recently, the impact of RW mutations was studied at two levels of KCNQ1 channels trafficking pathway: the early trafficking steps in the endoplasmic reticulum (ER) [18] and the endo/exocytosis recycling at the plasma membrane [30,31]. Using a combination of western blot and radioactive pulse chase experiments, we showed that three Nterminal RW mutations affect KCNQ1 proofreading by the cellular control quality ER-associated degradation (ERAD) system [18]. Indeed, the reduced expression levels observed for KCNQ1-Y111C, L114P and P117L mutant proteins with respect to WT KCNQ1 did not result from reduced rate of synthesis but from increased degradation in the proteasome. The “chaperoning role” of the ancillary KCNE1 subunit was directly addressed in this study. KCNE1 increased KCNQ1 stability and reduced its degradation in the proteasome, but this effect was not sufficient to restore normal trafficking of the mutant subunits. A differential role of KCNE1 in the plasma membrane endo/exocytosis cycling of KCNQ1 channel complex was recently documented by two studies from Seebohm et al. These authors first showed that KCNE1– KCNQ1 endocytosis and exocytosis involved Rab5 and Rab11 GTPases, respectively, and that the latter was controlled by the serum/ glucocorticoid-regulated kinase SGK1 [30]. More recently, they analyzed the differential regulation of Rab-dependent trafficking of KCNQ1 and KCNE1 mutants and showed that KCNQ1-Y111C, L114P and KCNE1-D76N mutations may be targeted to late endosomal compartment through Rab7-dependent vesicle trafficking [31]. 1.2.3. KCNQ1 gain-of-function mutations and KCNE1 KCNQ1 gain-of-function mutations have been also detected. They are associated with familial atrial fibrillation (AF) [32], short QT syndrome (SQT) – another cardiac arrhythmia increasing ventricular fibrillation susceptibility [33] – or with both phenotypes in the same

F. Charpentier et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 37–44

patient [34], all due to premature repolarization. When expressed with KCNE1, lone AF-associated S140G-KCNQ1 remains constitutively open in the voltage range of the AP in mammalian cells [32], resulting from a large (−57 mV) shift of the activation curve associated to a drastic slowing of deactivation kinetics as observed when expressed in Xenopus oocytes and stimulated at low frequencies [35]. On the other hand, the current generated by lone SQTassociated mutant V307L presents faster activation kinetics. Interestingly, the V141M mutation associated with the double phenotype induces both modifications. The finding that two adjacent mutations in KCNQ1 can convert slowly activating IKs channels into constitutively open channels suggests that interaction between the KCNE1 subunits and the extracellular end of the KCNQ1 S1 domain regulates activation kinetics. On the other hand, V307L mutation is located in the pore loop H5 and not only impairs activation but also abolishes inactivation when expressed alone [36]. G272 in S5 helix and V307 are suspected to interact and be responsible for inactivation during depolarization. Furthermore, KCNE1 possibly abolishes inactivation by weakening the interaction between H5 and S5 domains as suspected by Seebohm et al. [36]. 1.2.4. KCNE subunits: distribution and function More surprising is the report of a KCNQ1 mutation (Q147R) responsible for the simultaneous AF and LQT phenotype in the same patient [37]. The functional study of the mutant highlighted the physiological relevance in atria of KCNE2 (formerly called MiRP1), the second KCNE family member of K+ channel regulatory β-subunits to be cloned and primarily identified as an HERG regulator (see below) [38]. Unlike KCNE1, KCNE2 co-expression with KCNQ1 yields instantaneous currents with very low amplitude [39]. Despite very low mRNA levels in the human heart, KCNE2 is relatively more expressed in left atrium than in left and right ventricles [40]. When expressed in the presence of KCNE1, Q147R-KCNQ1 generated a reduced IKs-type current that correlates with depolarization delay at the ventricle level. In the presence of KCNE2, the mutated protein gave rise to currents presenting similar activation kinetics but greater amplitude than currents conducted by WT KCNQ1–KCNE2 channels, consistent with AF phenotype [40]. In the human atrium, predominant effect of KCNE2 over KCNE1 on Q147R-KCNQ1 is difficult to understand because of its low mRNA level. However, most studies to date have investigated the expression of KCNE subunits on the transcriptional level and little is known about the quantity of KCNE protein produced. Several members of the KCNE family are expressed in the heart. They have very different and specific effects on the complex they respectively form with KCNQ1 [40]. Surprisingly, the members of KCNE family with the lowest expression at the mRNA level seem to be involved in AF when mutated. This is the case of KCNE2 [41, and above] and KCNE5 [42]. On the other hand, common polymorphisms of the most expressed KCNE subunits KCNE1 [43] [44] and KCNE4 [45] have been shown to be frequent in AF patients. Finally, for the intermediately expressed KCNE3, a polymorphism co-segregated with AF but with no apparent effect on the mutant KCNE3 subunit function [46]. The respective role of all these regulating proteins in cardiac electrical activity remains to be understood. The pathophysiology of these mutations may depend on their differences in spatial distributions. The stoichiometry of the cardiac KCNQ1–KCNE complex also raised number of debates. Various methods of investigation resulted in variable protein ratio values. Using protein constructs containing one KCNE1 protein fused by its C-terminus to one to two KCNQ1 proteins, Wang et al. [47] proposed a stoichiometry of one to more than one KCNE1 per KCNQ1. On the other hand, using charybdotoxin-sensitive KCNQ1 and modified KCNE1 variants and studying charybdotoxindependent block level and kinetics, several studies resulted in a calculated KCNQ1:KCNE1 ratio of 4:2 [48,49]. This 4:2 ratio was

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confirmed by radioactive charybdotoxin and antibody binding assays [48]. Morin and Kobertz [50] demonstrated also the possibility of heterotetramers containing KCNE1 and KCNE4 or KCNE3 subunits. A complexity level was further introduced when it was proposed a hierarchical association affinity of KCNQ1 with the different KCNE subunits [50,51]. Using Kv channels crystal structure and experimental data on WT and mutant KCNQ1 and KCNE1, the structure of KCNQ1 subunits with KCNE1-binding pockets has been predicted [52–54]. Other KCNE subunits could interfere in the same pocket, and this interaction involves different KCNE amino acids located in the transmembrane segment and also C-terminus [55–57]. Finally to make things even more complicated, Tseng's team recently showed that there is a KCNE1 turnover in the sarcolemmal KCNQ1–KCNE1 complex and that it can be substituted by KCNE2, independently trafficking to the cell surface [58]. 1.2.5. KCNQ1 auto-regulation of channel trafficking In the heart, KCNQ1 encodes several isoforms, the pore-forming isoform 1 now called KCNQ1a, and isoform 2 (now KCNQ1b), differing in their NH2-termini [59,60]. We showed that when co-expressed in mammalian cells, KCNQ1b decreases the K+ current by retaining the pore isoform in the ER [28]. Most interestingly, KCNQ1b mRNA is relatively more abundant in the midmyocardium where IKs is the smallest and AP duration the longest when compared to endo- and epicardium [61]. Recently, Liu and collaborators [62] detected KCNQ1b protein and showed that its increased expression may contribute to IKs decrease and electrical remodeling in a canine model of ischemic cardiomyopathy. 1.3. IKs regulation 1.3.1. AKAPs and PKA-dependent regulation of IKs A field of research initiated in the late 1990s and still going is the track of channels' partner proteins. We mentioned above that through the PKA pathway, IKs plays a critical role in the control of the APD by catecholamines. Two PKA anchoring proteins have been suggested to interact with KCNQ1 and play a role in its regulation by PKA and phosphatases [29,63]. Interestingly, the presence of yotiao is not only necessary for the channel phosphorylation on Ser27 [29], and probably S468/T470 [64], but also for channel up-regulation once the channel has been phosphorylated [65]. The discovery that yotiao itself is phosphorylated brings in another level of complexity in the beta-adrenergic regulation of the IKs channel complex [66]. More recently, a phosphodiesterase has been shown to be also part of this channel complex [67]. This regulation has a pathophysiological relevance because KCNQ1 mutations that disrupt the KCNQ1–yotiao interaction have been identified (cf. above) as well as a mutation in yotiao itself, S1570L [68]. These mutations are clearly associated with, and most probably causing LQT. β-Tubulin has also been identified as an essential component for channel up-regulation once phosphorylated (and not for channel phosphorylation) [69]. 1.3.2. Calmodulin-dependent regulation IKs is also activated by intracellular Ca2+. A work in guinea pig cardiomyocytes suggested that Ca2+ sensitivity of IKs was due to the Ca2+-sensitive endothelial nitric oxide synthase NOS3 [70]. Another member identified in the IKs complex is calmodulin [71–73], suggesting that calmodulin may also directly modulate channel gating. Regulation by intracellular Ca2+ in cells expressing recombinant KCNQ1 and calmodulin has been observed in oocytes and HEK293 but not in CHO [70,71,74,75]. Calmodulin has been shown to be necessary to KCNQ1 membrane targeting [56,57] and required for the Snitrosylation of a cystein (C445) in KCNQ1 [74]. The S-nitrosylation of cystein 445 seems to partly mediate channel activation by NOS, suggesting that calmodulin is necessary in two steps of the NOS3 pathway: activation of NOS3 and S-nitrosylation of KCNQ1. It has also

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to be mentioned that NOS3 activation and subsequent IKs increase is also promoted by testosterone as suggested by the work of Bai and collaborators [76] on isolated guinea pig ventricular myocytes, and may contribute to the shorter QTc intervals measured in males. 1.3.3. Nedd4-2 and KCNQ1 degradation The Nedd4-2 ubiquitin–protein ligase also contributes to KCNQ1 complex. KCNQ1 contains a consensus PY motif interacting with Nedd4/Nedd4-like ubiquitin–protein ligases and Nedd4-2 coimmunoprecipitates with KCNQ1 in heterologous expression system. Moreover, expression of a dominant-negative Nedd4-2 led to an increase in IKs in adult guinea pig cardiomyocytes [77]. These results suggest that KCNQ1 internalization and stability are physiologically regulated by Nedd4/Nedd4-like-dependent ubiquitylation, which may regulate KCNQ1 channels surface density in cardiomyocytes. 1.3.4. PIP2 regulation of KCNQ1 Besides protein partners, lipids are also modulators of ion channels activity. The best documented example is the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) [78]. KCNQ1 and other KCNQ channels are up-regulated by PIP2 [79], a biophysical study on KCNQ1 suggesting a stabilization of the open state [80]. KCNQ1–PIP2 interaction is decreased by a least three LQT mutations targeting arginines, suggesting an interaction of this positively charged residues with PIP2 phosphates [81]. Alternatively, these mutations may alter the binding site conformation, as exemplified for KCNQ2 [82].

flows because of rapid inactivation of HERG channels. However, as the voltage becomes less positive, some channels recover from inactivation and pass current, leading to a progressive increase in repolarizing current, with maximal current occurring before the final rapid declining phase of the AP. These results have been confirmed by the elegant experiments of Berecki and co-workers[93] using dynamic AP clamp technique and by computer AP models. Mathematical models have also shown that although the maximum HERG current amplitude during an AP is relatively small compared to IK1, it contributes significantly to the initiation of the phase 3 repolarization of the action potential because it is activated at more positive potentials than those during which IK1 contributes the most [94]. AP clamp studies of native ventricular IKr have produced similar results [92,95–97]. The critical role played by channel inactivation in limiting HERG current during the early phases of the AP is further emphasized by the finding that an inactivation-attenuating HERG channel mutation leads to AP shortening and SQT [98,99]. 2.3. SQT1 mutations and inactivation

2. The fast component IKr

The first mutations responsible for the SQT syndrome (SQT1) were identified in KCNH2 [100]. Two different mutations were described, leading to the same amino acid change (N588K) in the S5-P loop region of HERG and resulting in a dramatic increase in IKr. Additional mutations have since been discovered, which all induce a gain of function of HERG channel [98,99]. All located in the outer mouth of HERG channels, namely the S5-P region, these mutations confirm the involvement of this domain in C-type inactivation [101].

2.1. Molecular identification of IKr-related channel, HERG

2.4. LQT2 mutations and trafficking

The molecular identity of the IKr K+ channel was unveiled after injection of the human ether-à-go-go-related gene (hERG or KCNH2) cRNA in Xenopus oocytes and the recording of IKr-like K+ currents [83,84]. A few weeks earlier, KCNH2 had been identified as involved in the LQT syndrome form 2 [85]. KCNH2 encodes a 6-transmembrane domains voltage-dependent K+ channel constituting the α-subunit of the IKr channel called HERG and more recently Kv11.1. It has been suggested that co-assembly of the regulatory β-subunit MiRP1 (minKrelated peptide 1 encoded by KCNE2 gene) with HERG α-subunits is required in order to reconstitute native IKr [38]. KCNE2 implication appeared important when gene variants were associated with congenital and drug-induced LQT [86,87]. However, this issue is controversial. Indeed, Weerapura et al. [88] reported that like the currents resulting from expression of HERG alone, those produced by HERG expressed with KCNE2 did not recapitulate native IKr properties neither. Moreover, there is strong evidence that KCNE2 actually regulates KCNQ1 channel (see above).

Numerous mutations in HERG account for the second most common form of LQT syndrome (LQT2 [83]). With the exception of the N629D mutation [102], HERG mutations characterized to date are loss-of-function mutations reducing IKr amplitude (via haplo-insufficiency or dominant-negative suppression of WT channels) and consecutively prolonging cardiac repolarization [103]. Failure of cell surface expression because of defective HERG trafficking is known to cause LQT2 in some cases. Misfolded and incompletely assembled proteins are common side products of ER protein synthesis, and the cellular quality control recognizes such defects and results in their retention in the ER. To date, few ER-associated chaperones and adaptors have been shown to assist HERG protein folding and its trafficking in the secretory pathway. Those include HSP70, HSP90 [104], calnexin [105], 14-3-3 [106], Sar1 and ARF1 GTPases [107]. In addition, it was shown that the Golgi-associated protein GM130 interacts with HERG C-terminus as the channel is transported between the ER and the plasma membrane in the Golgi apparatus [108]. This interaction facilitates the channel trafficking and it is disrupted by specific LQT2 mutations. HERG trafficking rapidly became a focus of interest for three reasons: (1) most of its mutations give rise to trafficking-deficient channel proteins; (2) the trafficking of these mutants can be restored by high-affinity HERG channel-blocking drugs and the channels are still capable of conducting IKr current when intracellular trafficking is restored. For example, Rajamani et al. [109] showed that fexofenadine rescued the electrophysiologic defect without complete channel blockade, suggesting that this might be a useful treatment for some LQT2 patients. (3) More recently, a few pharmaceutical compounds were shown to specifically block HERG channel trafficking [110–112]. Deficient trafficking HERG mutations and their rescue by pharmacological agents were presented in excellent reviews [113,114]. Here we will focus on acquired forms of LQT (acLQT) that affect channel trafficking. AcLQT has been previously identified to result from pharmacological interventions, often for the purpose of treating diseases unrelated to cardiac dysfunction. For instance, antihistaminics,

2.2. Uniqueness of IKr among voltage-dependent K+ currents The current generated by HERG channels shows unusual voltage dependence. Under conventional voltage clamp, HERG current increases with potential above −40 mV – as do other voltage-gated channels – but declines with depolarizations above 0 mV. The amplitude of tail currents on repolarization exceeds that of currents during the depolarizing pulse [83,84]. The unusual voltage dependence of HERG current results from a fast, voltage-dependent C-type inactivation process, which limits K+ flow at positive voltages [89,90]. The large tail currents on repolarization from positive voltages are due to rapid recovery of inactivated channels into a conducting state. The physiological consequences of HERG rapid voltage-dependent inactivation during depolarization and rapid recovery from inactivation during repolarization have been analyzed under AP voltage clamp of mammalian cell lines expressing recombinant HERG channels [91,92]. During the plateau of the ventricular AP, little HERG current

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antipsychotics or antibiotics can predispose patients to lethal arrhythmias [115]. The common property of these drugs is that they decrease IKr by depressing HERG channel gating. In fact, the unusual susceptibility of HERG channel to blockade by drugs in comparison with other voltagegated K+ channels may be partially explained by the specific presence of aromatic residues in the channel pore which harbors this unique drugbinding site [116,117]. Recently, a novel mechanism for acLQT has emerged, which involves compounds interfering with HERG trafficking. These include arsenic trioxide [110], pentamidine [111], probucol (a cholesterol lowering therapeutic compound) [118] and cardiac glycosides [112] (for review, see [119]). Whereas arsenic was shown to interfere with HERG trafficking probably by inhibiting channel–HSP90 interactions, recent studies by Ficker's group unveiled a new peculiar mechanism for cardiac glycosides effect. In a first report, they showed that inhibition of HERG trafficking by cardiac glycosides results from direct inhibition of Na+/K+-ATPase but not from off-target direct interaction with HERG [35]. In a more recent study, they unveiled the crucial role of the cellular K+ depletion resulting from Na+/K+-ATPase inhibition in the disruption of HERG trafficking [120]. Interestingly, they showed that intracellular K+ depletion effects were reversed by mutations on the aromatic residues discussed above and located in the channel pore. Together their data are compatible with a localized structural rearrangement in HERG selectivity filter during biosynthesis in low [K+]I condition that induces misfolding and ER retention of the channel protein. 2.5. HERG1a/1b and trafficking As for KCNQ1, several studies suggest that native IKr channels are heteromers arising from co-assembly of two different HERG isoforms: HERG1a (or HERG) with HERG1b, another α subunit encoded by an alternate transcript of KCNH2 with a unique N terminus [121]. Sale et al. [122] characterized the heterotetramers and showed that they generate a greater current than HERG1a expressed alone. This is facilitated by direct, cotranslational interactions of the divergent N termini that mask an exposed ER retention signal (RXR) in the HERG1b subunit, which otherwise prevents efficient expression of hERG1b homomeric channels [123]. These investigations showed also heterotetramers increased occupancy of the open state. In addition, HERG1a/1b channels develop E-4031 block with slower kinetics and reduced sensitivity. Finally, they described an HERG1b-specific A8V missense mutation in LQT2 patients. Mutant HERG1b A8V expressed alone or with HERG1a in HEK-293 cells dramatically reduced HERG1b protein levels. This further supports the physiological role of alternative HERG isoform. 2.6. HERG partners and IKr regulation 2.6.1. KCR1 and HERG pharmacological response The membrane protein KCR1 was first shown to facilitate functional expression of neuronal non-inactivating K+ currents associated with rat EAG voltage-dependent K+ channels that have a high homology with HERG [124]. KCR1 also accelerates their activation. Far-Western blotting revealed that the proteins interacted through their C-terminal regions. In the human heart, KCR1 can be immunoprecipitated with HERG [125]. Functionally, KCR1 was shown to reduce the sensitivity of HERG to classic proarrhythmic HERG blockers [125]. Except this important role in cardiac electropharmacology, the physiological or pathophysiological role of this new partner in HERG function remains to be determined. 2.6.2. ARHGAP6, PIP2 and HERG ARHGAP6 (rho-GTPase activating protein 6) has been detected as potential HERG partner in a very original model for cardiac electrophysiology: Caenorhabditis elegans [126]. In this model, Potet et al. [126] combined RNA interference with behavioral screening to

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detect genes that influence function of the HERG C. elegans homolog, UNC-103. They observed that ARHGAP6 reduced membrane expression of HERG independently of its GTPase activity. Deciphering the regulation mechanism, they showed that ARHGAP6 reduces PIP2 levels by enhancing the enzymatic activity of a phospholipase C, PLCδ1. This is consistent with the data of Bian et al. [127] who demonstrated that HERG current amplitude and kinetics are sensitive to PLC-dependent PIP2 levels. Thus, enhanced PLC activity caused by ARHGAP6 is a required intermediary that leads to decreased PIP2 levels, which then negatively influence HERG current amplitude. In addition, the ARHGAP6–PLC pathway observed in Potet's study affects the cytoskeletal architecture with possible negative effects on HERG trafficking. Finally, the consensus SH3 binding domain located in HERG C-terminus seems to be involved in HERG–ARHGAP6 interplay. However, as for KCR1, ARHGAP6 physiological or pathophysiological role of this protein in HERG function remains to be determined. 3. KCNQ1 and HERG are partners IKr and IKs are functionally linked; when IKr is reduced, the action potential is prolonged, causing IKs activation to increase so as to prevent excess repolarization delay [128]. However, a new degree of complexity was reached when it was suggested that IKr and IKs channel complexes might also be structurally linked. Ehrlich et al. [129] demonstrated that molecular interactions exist between the HERG and the KCNQ1 α-subunits. Using a combination of biophysical and biochemical techniques, the authors showed that KCNQ1 can interact with and modify the localization and increase the currentcarrying properties of HERG. Very recently, they determined that an LQT-related KCNQ1 mutation, T587M, which has previously been associated with a severe clinical phenotype, affects this interaction [130]. KCNQ1–T587M failed to increase HERG-dependent K+ current highlighting the role of KCNQ1 A-domain in the interaction mechanisms (see above). It also unveils a novel mechanism by which KCNQ1 mutations may produce LQT2-type clinical manifestations. References [1] Noble D, Tsien RW. Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres. J Physiol 1969;200:205–31. [2] Noble D, Tsien RW. The kinetics and rectifier properties of the slow potassium current in cardiac Purkinje fibres. J Physiol 1968;195:185–214. [3] Sanguinetti MC, Jurkiewicz NK. Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 1990;96:195–215. [4] Lu Z, Kamiya K, Opthof T, Yasui K, Kodama I. Density and kinetics of IKr and IKs in guinea pig and rabbit ventricular myocytes explain different efficacy of IKs blockade at high heart rate in guinea pig and rabbit: implications for arrhythmogenesis in humans. Circulation 2001;104:951–6. [5] Jost N, Virág L, Bitay M, Takács J, Lengyel C, Biliczki P, Nagy Z, Bogáts G, Lathrop DA, Papp JG, Varró A. Restricting excessive cardiac action potential and QT prolongation: a vital role for IKs in human ventricular muscle. Circulation 2005;112:1392–9. [6] Jurkiewicz NK, Sanguinetti MC. Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III antiarrhythmic agent. Specific block of rapidly activating delayed rectifier K+ current by dofetilide. Circ Res 1993;72: 75–83. [7] Walsh KB, Kass RS. Regulation of a heart potassium channel by protein kinase A and C. Science 1988;242:67–9. [8] Sanguinetti MC, Jurkiewicz NK, Scott A, Siegl PK. Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action. Circ Res 1991;68:77–84. [9] Walsh KB, Kass RS. Distinct voltage-dependent regulation of a heart delayed IK by protein kinases A and C. Am J Physiol 1991;261:C1081–90. [10] Van Wagoner DR, Kirian M, Lamorgese M. Phenylephrine suppresses outward K+ currents in rat atrial myocytes. Am J Physiol 1996;271:H937–46. [11] Cui J, Melman Y, Palma E, Fishman GI, McDonald TV. Cyclic AMP regulates the HERG K+ channel by dual pathways. Curr Biol 2000;10:671–4. [12] Takumi T, Ohkubo H, Nakanishi S. Cloning of a membrane protein that induces a slow voltage-gated potassium current. Science 1988;242:1042–5. [13] Hausdorff SF, Goldstein SA, Rushin EE, Miller C. Functional characterization of a minimal K+ channel expressed from a synthetic gene. Biochemistry 1991;30: 3341–6. [14] Wang Q, Curran ME, Splawski I, Burn TC, Millholland JM, VanRaay TJ, Shen J, Timothy KW, Vincent GM, de Jager T, Schwartz PJ, Toubin JA, Moss AJ, Atkinson

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