Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2

BBRC Biochemical and Biophysical Research Communications 344 (2006) 814–820 www.elsevier.com/locate/ybbrc

Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2 Futoshi Toyoda b

a,*

, Hisao Ueyama b, Wei-Guang Ding a, Hiroshi Matsuura

a

a Department of Physiology, Shiga University of Medical Science, Seta-Tsukinowa, Otsu, Shiga 520-2192, Japan Department of Molecular Medical Biochemistry, Shiga University of Medical Science, Seta-Tsukinowa, Otsu, Shiga 520-2192, Japan

Received 27 March 2006 Available online 19 April 2006

Abstract The KCNE proteins (KCNE1 through KCNE5) function as b-subunits of several voltage-gated K+ channels. Assembly of KCNQ1 K+ channel a-subunits and KCNE1 underlies cardiac IKs, while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch–clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.  2006 Elsevier Inc. All rights reserved. Keywords: IKs; KCNE1; KCNE2; KCNQ1; Mefenamic acid; Long QT syndrome; K+ channel; Heart; Patch–clamp

The KCNE protein (encoded by five genes designated KCNE1 through KCNE5) is a family of single transmembrane peptides (103–177 residues in length) that function as ancillary b-subunits of several voltage-gated K+ channels [1]. Functional coupling of KCNE1 with KCNQ1 K+ channel to recapitulate many properties of the slowly activating and deactivating cardiac delayed rectifier K+ current (IKs) is well documented [2,3]. Mutations in either the KCNE1 or KCNQ1 gene have been implicated in inherited long QT syndrome (LQTS) [4]. On the other hand, KCNQ1 is also found to have an affinity for all other members of the KCNE protein, forming K+ channels with different voltage-dependence of activation, gating properties, and pharmacology [5–8]. Recent real-time PCR experiments have revealed substantial expression of gene tran-

*

Corresponding author. Fax: +81 77 548 2348. E-mail address: [email protected] (F. Toyoda).

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.213

scripts for all KCNE subunits in the heart [9,10]. However, contribution of multiple KCNE subunits to IKs regulation remains to be determined. KCNE2 is first described as a regulatory protein of HERG channel to form a rapid component of cardiac delayed rectifier K+ current (IKr) [11]. Until now, functional associations with a number of ion channel a-subunits including KCNQ1 [6], Kv4.2 [12], and HCN1 [13] have been revealed. Importance of KCNE2 in cardiac electrical activity is evident from the fact that mutations in the KCNE2 gene have been found in cases of LQTS and atrial fibrillation [11,14]. Nevertheless, its physiological relevance to cardiac ionic currents is still in debate. Our main interest is to elucidate the involvement of KCNE2 in regulation of cardiac IKs channel. The present study was designed to investigate whether KCNE2 can exert functional regulation of KCNQ1 channels in the presence of KCNE1. In this paper, we demonstrate collaborative effects of KCNE1 and KCNE2 on electrophysiological

F. Toyoda et al. / Biochemical and Biophysical Research Communications 344 (2006) 814–820

and pharmacological properties of KCNQ1 channels. Possible formation of heteromultimeric complexes will be discussed. Materials and methods Cloning of KCNE gene and transfection. Human KCNE1 and KCNE2 cDNA was obtained by PCR from human heart cDNA library (Clontech) and cloned into PCR3.1 mammalian expression vector (Invitrogen). Human KCNQ1 subcloned into PCI was a gift from Dr. J. Barhanin (IPMC, CNRS, France). Plasmid DNA of the KCNE1–KCNQ1 and KCNE2–KCNQ1 fusion protein was constructed by insertion of KCNE1 and KCNE2, respectively, into PCI-KCNQ1 plasmid. At first, KCNE1 and KCNE2 DNA in which the C terminus of each KCNE (stop codon omitted) was followed by a short sequence of the N terminus of KCNQ1 (10 bp) was amplified with PCR using the following downstream primers: 5 0 -CCGCGGCCATTGGGGAAGGCTTCGTCTC-3 0 and 5 0 -CCGC GGCCATGGGGGACATTTTGAACCC-3 0 for KCNE1 and KCNE2, respectively (underline indicates the SacII recognition sequence). The PCR products were digested with SacII (Takara) and then ligated by a restriction fragment of 240 bp in KCNQ1 with NotI (Takara) and SacII. Finally, these chimeric sequences were inserted into PCI-KCNQ1plasmid DNA truncated between NheI site under T7 promotor and NotI site in coding region. COS7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (GIBCO) and antibiotics (100 U/ ml penicillin and 100 lg/ml streptomycin) in a humidified incubator gassed with 5% CO2 and 95% air at 37 C. Before transfection, cells were seeded onto 35-mm plastic culture dishes with 7–8 glass coverslips (5 mm · 3 mm) bottom and incubated for 24–48 h. Transient transfection was performed using Lipofectamine (Invitrogen). The amounts of each cDNA used for transfection were (mg/dish): 0.75 KCNQ1, 0.75 KCNE1, 0.75 KCNE2, and 0.5 GFP (1.0 KCNE1–KCNQ1 or KCNE2–KCNQ1, 1.0 KCNE2 or KCNE1 and 0.5 GFP for the experiment using fusion constructs). Forty-eight to seventy-two hours after transfection, only GFP-positive cell was used for the patch–clamp study. Preparation of guinea-pig ventricular cells. All animal experiments were approved by the institution’s Animal Care and Use Committee and were performed in accordance with The Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication number 85-23, revised 1996). Guinea-pig ventricular cardiomyocytes were isolated by the enzymatic dissociation procedure. Briefly, hearts were quickly excised from female guinea-pigs (250–350 g) deeply anaesthetized by intraperitoneal injection of sodium pentobarbital (120 mg/kg B.W.) and mounted on a Langendorff apparatus. Isolated hearts were retrogradely perfused via aorta at 37 C, initially for 4 min with normal Tyrode solution, then for 4 min with nominally Ca2+-free Tyrode solution prepared by omitting CaCl2 from the normal Tyrode solution, and finally for 8–12 min with nominally Ca2+-free Tyrode solution containing 0.4 mg/ml collagenase (Wako, Osaka, Japan). Thereafter, ventricular tissue was cut into small pieces and mechanically dissociated in high-K+, low-Cl (KB) solution. The isolated cells were then stored at 4 C in KB solution for later use. Patch–clamp recordings. Whole-cell membrane current was recorded using the whole-cell configuration of the patch–clamp technique with an EPC-8 patch–clamp amplifier (HEKA). A coverslip with adherent COS7 cells or a small aliquot of ventricular cell-containing suspension was placed on the glass bottom of a recording chamber (0.5 ml in volume) mounted on the stage of an inverted microscope (TMD-300, Nikon). Patch–clamp pipettes were prepared from glass capillary tube (Narishige) by means of a horizontal pipette puller (P-97, Sutter Instrument), and the tips were then fire-polished with a microforge (MF-83, Narishige). Pipette resistance was 2–4 MX when filled with internal solution. Current recordings were conducted at 34 ± 1 C. Currents and voltages were digitized and voltage commands were generated through a LIH-1600 AD/DA interface (HEKA) controlled by PatchMaster software (Version 2.03, HEKA).

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Data analysis. Membrane capacitance (Cm) was calculated by fitting a single exponential function to the decay phase of the transient capacitive current in response to ±5 mV voltage steps (20 ms) from a holding potential of 50 mV. Current amplitudes were divided by Cm to obtain current densities (pA/pF). The voltage-dependence of current activation was determined by fitting the normalized tail current (Itail) versus test potential (Vtest) to a Boltzmann function expressed by: Itail = 1/ (1 + exp[(V0.5  Vt)/k]), where V0.5 is the voltage at which the current is half-activated and k is the slope factor. Time constants for deactivation (sfast and sslow) were obtained by fitting a two-exponential function to the time course of deactivating tail currents. All data were expressed as means ± SEM. Statistical comparisons were made using ANOVA, followed by Tukey test, and differences were considered significant at p < 0.05. Solutions and drugs. External Tyrode solution contained (mM): 140 NaCl, 0.33 NaH2PO4, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 5.4 glucose, and 5 Hepes, and pH was adjusted to 7.4 with NaOH. In experiments using ventricular myocytes, test solution for IKs current recording was made by adding 0.4 lM nisoldipine (as 1 mM stock solution in ethanol) and 0.5 lM E-4031 (as 5 mM stock solution in distilled water) to the external solution to eliminate ICa,L and IKr, respectively. The internal pipette solution contained (mM): 70 potassium aspartate, 50 KCl, 10 KH2PO4, 1 MgCl2, 3 Na2-ATP, 0.1 Li2-GTP, 5 EGTA, and 5 Hepes, and pH was adjusted to 7.2 with KOH. Liquid junction potential between the test solution and the pipette solution was measured to be around 10 mV and was corrected. Chromanol 293B and mefenamic acid were added from 0.1 M stock solution in DMSO to the external solution (final DMSO concentration did not exceed 0.1%).

Results Synergic effects of KCNE1 and KCNE2 on KCNQ1 channels To investigate properties of currents resulting from coexpression of KCNQ1 with KCNE1, with KCNE2 or with both of KCNE1 and KCNE2, equal amounts of each plasmid DNA were transiently co-transfected into COS7 cells. Consistent with previous studies [2,3], co-expression of KCNQ1 with KCNE1 (Figs. 1A and D) produced slowly activating outward current in response to depolarizing voltage steps to potentials more than 40 mV from a holding potential of 80 mV and then slowly decaying tail current upon hyperpolarizations to 50 mV. In contrast, the association with KCNE2 (Figs. 1B and D) gave rise to voltage- and time-independent instantaneous current that increased linearly with voltage and reversed in direction at potential of 82.1 ± 1.3 (n = 6), closed to EK (84 mV as predicted by Nernst equation). Thus, electrophysiological properties of the KCNQ1/KCNE1 and KCNQ1/KCNE2 currents were clearly distinguishable, yet both currents were sensitive to chromanol 293B, a KCNQ1 channel blocker. Bath application 0.1 mM 293B unmasked small background leak (Figs. 1A and B). Net current density (the step current amplitude reduced by 293B was normalized with cell membrane capacitance) measured at +30 mV showed that coupling of KCNQ1 with KCNE1 produced current five times larger than with KCNE2 (Fig. 1D). Unexpectedly, when both KCNE1 and KCNE2 were co-expressed with KCNQ1, current recordings appeared never to be an ensemble of the KCNQ1/

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Fig. 1. Whole-cell membrane currents recorded from COS7 cells transfected with KCNQ1 and KCNE1 (A, Q1 + E1), KCNQ1 and KCNE2 (B, Q1 + E2), and KCNQ1, KCNE1, and KCNE2 (C, Q1 + E1 + E2) cDNA. Currents were elicited by 4 s (A,C) or 1 s (B) voltage steps from a holding potential of 80 mV over the range of 110 to +50 mV in 10 mV increments before (left) and after (right) application of 0.1 mM 293B. Tail currents were recorded upon a repolarization step to 50 mV. Dotted line indicates zero level. (D) Bar graph of averaged net current density summarized for timedependent and instantaneous currents during voltage-step to +30 mV. Net current amplitude, totally blocked by 0.1 mM 293B, was normalized by cell membrane capacitance (Cm). Instantaneous current level was measured at 5 ms after depolarization. Time-dependent current was estimated as a difference between a current level at the end of depolarizing step and an instantaneous current level. (E) Voltage-dependence of activation in the KCNQ1/KCNE1 and KCNQ1/KCNE1/KCNE2 currents. Tail current amplitude normalized by a value obtained at +50 mV was averaged against each test potential and fitted by Boltzmann equation. (F) Bar graph of averaged sfast, sslow, and Afast/(Afast + Aslow) for the KCNQ1/KCNE1 and KCNQ1/KCNE1/KCNE2 currents.

KCNE1 and KCNQ1/KCNE2 currents (Fig. 1C). As is evident in Fig. 1D, current was almost totally time-dependent and its magnitude was comparable to that of the KCNQ1/KCNE1 current, implying dominant effects of KCNE1. Although the appearance of currents obtained by co-expression of KCNQ1 with both KCNE1 and KCNE2 resembled the KCNQ1/KCNE1 current, quantitative analysis revealed several differences in biophysical properties. Fig. 1E illustrates the voltage-dependence of activation where the tail current amplitude at each test potential was normalized with reference to its maximal value at +50 mV and was then fitted by Boltzmann equation. The V0.5 for the KCNQ1/KCNE1/KCNE2 current was significantly shifted towards depolarizing potentials (+6.6 ± 1.1 mV, n = 14), compared to that for the KCNQ1/KCNE1 current (2.3 ± 2.4 mV, n = 7, p < 0.01). There was no difference in the value of k (13.8 ± 0.4 and 14.1 ± 0.6 mV for the KCNQ1/KCNE1/ KCNE2 and KCNQ1/KCNE1 currents, respectively). Furthermore, differences between the KCNQ1/KCNE1/ KCNE2 and KCNQ1/KCN1 currents were also manifested in the time course of deactivation (Fig. 1F). By fitting two-exponential function to the tail current decay at 50 mV, the time constants of the fast component (sfast) and of the slow component (sslow) were determined to be 141 ± 9 and 404 ± 41 ms (n = 22), respectively, for the KCNQ1/KCNE1/KCNE2 current, and these values were significantly smaller than corresponding values for the

KCNQ1/KCNE1 current (sfast, 203 ± 11 ms; sslow, 649 ± 53 ms; n = 18). These results suggest that KCNE2, in addition to KCNE1, can participate in synergic control of KCNQ1 channels. Functional association of KCNE2–KCNQ1 fusion channels with KCNE1 We next constructed a plasmid encoding a fusion protein in which the N terminus of KCNQ1 was fused to the C terminus of either KCNE1 or KCNE2, and characterized the function of heteromeric channels by measuring whole-cell currents. The use of these fusion constructs was validated by checking that the fusion channels produced currents with properties similar to those obtained by co-expression of KCNQ1 with KCNE1 or KCNE2. As is expected from previous works [15,16], expression of KCNE1–KCNQ1 fusion proteins could successfully form slowly activating K+ channels that had V0.5 of +1.3 ± 3.1 mV (n = 9) and deactivation time constants (sfast, 190 ± 14 ms; sslow, 584 ± 60 ms; n = 14), very similar to those obtained by co-expression of KCNE1 and KCNQ1. Likewise, as demonstrated in Fig. 2A, the KCNE2–KCNQ1 fusion channels evoked instantaneous currents, again similar to those induced by co-expression of KCNE2 and KCNQ1. We, then, co-expressed each fusion construct with either KCNE1 or KCNE2 with a 1:1 DNA ratio to exploit whether functional channels composed of KCNQ1, KCNE1, and KCNE2 can be formed.

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Fig. 2. Representative current traces recorded from COS7 cells expressing KCNE2–KCNQ1 fusion construct alone (A, E2-Q1) or KCNE2–KCNQ1 and KCNE1 (B, E2-Q1 + E1). Currents were elicited using 1 s (A) or 4 s (B) voltage step before (left) and after (right) application of 0.1 mM 293B. (C) Bar graph of averaged net current density summarized for time-dependent and instantaneous components in the KCNE2–KCNQ1 and KCNE2–KCNQ1/ KCNE1 currents during voltage-step to +30 mV. (D,E) Voltage-dependence of activation and bar graph of averaged sfast, sslow and Afast/(Afast + Aslow) in the KCNE1–KCNQ1, KCNE1–KCNQ1/KCNE2, and KCNE2–KCNQ1/KCNE1 currents.

As demonstrated in Fig. 2B, coupling of the KCNE2– KCNQ1 with KCNE1 exclusively produced large voltageand time-dependent current without obvious instantaneous component, providing a functional evidence to show that all KCNE2–KCNQ1 co-assembles with KCNE1. In further support of the view, the KCNE2–KCNQ1/KCNE1 current had more depolarized V0.5 (+9.4 ± 2.1 mV, n = 7, p < 0.05) and faster deactivation kinetics (sfast, 149 ± 24 ms; sslow, 365 ± 41 ms; n = 5, p < 0.05), compared to the KCNE1–KCNQ1 current (Figs. 2D and E). In comparison with current obtained by co-expression of KCNE1–KCNQ1 with KCNE2 (V0.5, +6.5 ± 4.2 mV, n = 4; sfast, 163 ± 11 ms; sslow, 389 ± 71 ms; n = 7), however, there was no difference in these parameters. The role of KCNE proteins on sensitivity to mefenamic acid Mefenamic acid is a nonsteroidal anti-inflammatory drug and is known to affect a variety of ion channels, including the KCNQ1/KCNE1 channels heterologously expressed in Xenopus oocyte and CHO cells [17–19]. In the present study, we found mefenamic acid to be a good probe for discrimination of the KCNQ1/KCNE1 and KCNQ1/KCNE1/KCNE2 channels. In Fig. 3, we compared the effects of mefenamic acid on current recordings obtained by co-expression of KCNQ1 with KCNE1, with KCNE2 or with both of KCNE1 and KCNE2. Consistent with previous observations [17–19], the KCNQ1/KCNE1 current was dramatically affected by bath application of 0.1 mM mefenamic acid, which was characterized by marked inhibition of tail current decay, thereby increasing instantaneous current in response to voltage-steps

(Fig. 3A). Further addition of 0.1 mM 293B almost completely abolished the outward current except for background leak current, implying that the current changes during exposure to mefenamic acid were due to a modification of the KCNQ1/KCNE1 current. On the other hand, the KCNQ1/KCNE2 current was minimally affected by mefenamic acid (Fig. 3B), suggesting that response of current to mefenamic acid was highly dependent on KCNE subunits coupling to KCNQ1. When co-expressed with both KCNE1 and KCNE2, current gave response apparently different from that of the KCNQ1/KCNE1 current (Fig. 3C). In the presence of mefenamic acid, gating kinetics of the KCNQ1/KCNE1/KCNE2 current remained intact with an exception of modest retardation in tail current. In Fig. 4, the effects of mefenamic acid on deactivation time course were compared between the KCNQ1/ KCNE1 and KCNQ1/KCNE1/KCNE2 current. Both currents were increased in peak amplitude by 1.3- to 1.4-fold after application of mefenamic acid. However, mefenamic acid stabilized 71.6 ± 3.5% (n = 8) of the KCNQ1/KCNE1 channels in its open state even 4 s after recording at 50 mV, while only 26.4 ± 4.2% (p < 0.01, n = 6) of the KCNQ1/KCNE1/KCNE2 channels remained to be opened at the same point. The effects of mefenamic acid on cardiac IKs Finally, we sought to examine the responses of cardiac IKs to mefenamic acid in guinea-pig ventricular myocytes. Fig. 5A shows superimposed current traces in response to depolarizing pulses to membrane potentials of 40 to +50 mV in 10 mV steps applied from a holding potential

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Fig. 3. The KCNQ1/KCNE1 (A), KCNQ1/KCNE2 (B), and KCNQ1/KCNE1/KCNE2 (C) currents in control condition (left), during bath application of 0.1 mM mefenamic acid (middle) and further addition of 0.1 mM 293B (right). Each current was elicited with same voltage protocol in Fig. 1.

B

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Q1 + E1 + E2

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Fig. 4. Effects of mefenamic acid on deactivation time course in the KCNQ1/KCNE1 (A) and KCNQ1/KCNE1/KCNE2 (B) currents. Tail currents in control condition (black) and in the presence of mefenamic acid (grey) were normalized by peak amplitude in control and then averaged in several experiments.

of 50 mV. Under condition in which IKr and ICa,L were blocked by 0.5 lM E-4031 and 0.4 lM nisoldipine, respectively, the slowly activating outward current during the depolarizing pulses to potentials above 20 mV and the tail current evoked upon return to the holding potential were most reasonably attributed to IKs. Indeed, bath application of chromanol 293B almost completely abolished the time-dependent outward current without affecting large instantaneous current jump due to contamination of the inward rectifying K+ current (IK1) (Fig. 5A, right). External application of mefenamic acid substantially enhanced IKs (Fig. 5A, middle). As illustrated in Fig. 5B, the tail current amplitude was increased by 24.9 ± 4.8% (n = 3) in the presence of mefenamic acid. However, the rate of tail current decay was still fast enough to deactivate channels

almost completely within 4-s recording at 50 mV. Thus, at least in sensitivity to mefenamic acid, cardiac IKs channels appeared to be close to the KCNQ1/KCNE1/KCNE2 channels rather than the KCNQ1/KCNE1 channels. Discussion The effects of multiple KCNE subunits on KCNQ1 channels have been of recent interest [9,10]. Earlier observations indicate that even in the presence of KCNE1 other member of KCNE subunits can exert gating control of KCNQ1 channels [10,20], yet there is no evidence for the heteromultimerization of KCNQ1, KCNE1, and another KCNE subunit. In this article, we have demonstrated the synergic effects of KCNE1 and KCNE2 on electrophysio-

F. Toyoda et al. / Biochemical and Biophysical Research Communications 344 (2006) 814–820

A

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Fig. 5. (A) Native IKs recorded from guinea-pig ventricular myocyte in control condition (left), during bath application of 0.1 mM mefenamic acid (middle) and further addition of 0.1 mM 293B (right). Cell was held at 50 mV and depolarized to +50 mV for 4 s in 10 mV steps. (B) Effects of mefenamic acid on deactivation time course of IKs in guinea-pig ventricular myocytes.

logical and pharmacological properties of KCNQ1 channel, providing the first evidence to suggest that both subunits can be functionally associated with a KCNQ1 channel molecule forming heteromultimeric complexes with unique current properties. It was previously reported that co-expression of KCNQ1 with KCNE1 and KCNE3 resulted in a linear summation of the KCNQ1/KCNE1 and KCNQ1/KCNE3 current [5], whereas the dominant effect of KCNE3 was also reported [10,20]. These findings suggest that KCNQ1 is unable to generate heteromultimers with KCNE1 and KCNE3, in contrast to our observations. Recent studies have proposed that KCNE1 and KCNE3 exert their distinct effects on gating behavior of KCNQ1 channels through a structurally common mechanism. In a series of works by Melmann et al. [21,22], a single amino acid within the transmembrane segment (T58 and V72 in KCNE1 and KCNE3, respectively) was identified as a particular position for the specific modulation of KCNQ1 channel. Subsequent study demonstrated physical interaction of these amino acids with pore-lining region at residues 338–340 of KCNQ1 channel [23]. Thus, KCNE1 and KCNE3 appear to share a same region in KCNQ1 in a competitive manner. It is not known whether the corresponding amino acid (I64) in KCNE2 is a specific position for the regulation of KCNQ1 channel, although a mutation at other position (I57T) also causes drastic change in gating kinetics of the KCNQ1/KCNE2 channel [6]. Furthermore, in the present

study, a fixed 4:4 stoichiometry for KCNQ1-KCNE2 (or KCNQ1-KCNE1) binding with fusion constructs did not prevent additional effects of co-expressed KCNE1 (or KCNE2) (Fig. 2). Taken together, it is likely that KCNE2 utilizes a different mechanism for coupling to KCNQ1. In the present study, we have not confirmed physical association of KCNQ1, KCNE1, and KCNE2. Previous co-immunoprecipitation experiment showed that, when KCNQ1, KCNE1, and KCNE2, synthesized by in vitro translation with rabbit reticulocyte lysate, were incubated, both KCNE subunits were co-precipitated with KCNQ1 with equivalent affinity [6]. This observation may not be alone sufficient for indicating the heteromultimerization of KCNQ1, KCNE1, and KCNE2, although our current recordings exclude the possibility of forming distinct KCNQ1/KCNE1 or KCNQ1/KCNE2 channels. It is now generally accepted that the IKs channel is formed by the co-assembly of KCNQ1 and KCNE1 [2,3]. In the present study, however, we demonstrated differential effects of mefenamic acid on native IKs and recombinant KCNQ1/KCNE1 channels. Furthermore, additional expression of KCNE2 attenuated the sensitivity to mefenamic acid to some extent closed to the response of native IKs, suggesting that KCNE2 may be an important cofactor regulating cardiac IKs channel. Despite the physiological relevance of the KCNQ1/KCNE2 channels to background K+ conductance in acid secretory gastric parietal cells [24], the role of KCNE2 in the heart is now in debate due to its

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low expression level [9,10,25]. Thus, we could not deny limited contribution of KCNE2 to IKs regulation in vivo. The expression of KCNE2 is heterogeneous within different regions of a heart: higher density in atrial pacemaker tissue and Purkinje fibers compared to ventricular muscle [10,25,26] and displays developmental change or pathological change in diseased hearts [27]. Thus, involvement of KCNE2, albeit partially, may be important to generate functional diversity of IKs channels in particular circumstances. Several missense mutations and polymorphisms have been identified from the KCNE2 gene in patients with LQTS or atrial fibrillation [11,14]. It was recently shown that mutations in KCNE2 had gain-of-function or loss-offunction effects on the KCNQ1/KCNE2 channels [14]. Further studies are needed to elucidate the contribution of KCNE2 to regulate cardiac IKs channel and the involvement of its mutations in electrical abnormalities in inherited cardiac disease.

[11]

[12]

[13]

[14]

[15]

Acknowledgments [16]

This study was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science. We thank Drs. Hans-J. Lang and Ju¨rgen Pu¨nter (Aventis Pharma Deutschland GmbH) for kindly providing the chromanol 293B.

[17]

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