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Am J Physiol Heart Circ Physiol 286: H1300-H1309, 2004. First published December 11, 2003; doi:10.1152/ajpheart.00344.2003
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Subcellular localization of the delayed rectifier K+ channels KCNQ1 and ERG1 in the rat heart

Hanne Borger Rasmussen,1 Morten Møller,2 Hans-Günther Knaus,3 Bo Skaaning Jensen,4 Søren-Peter Olesen,1 and Nanna Koschmieder Jørgensen1

1Department of Medical Physiology and Copenhagen Heart Research Center, and 2Institute of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark; 3Institute of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria; and 4Division of Ion Channel Pharmacology, NeuroSearch, DK-2750 Ballerup, Denmark

Submitted 11 April 2003 ; accepted in final form 3 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the heart, several K+ channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K+ currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K+ channels, KCNQ1 and ether-a-go-go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter. Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these K+ channels to the transverse tubular system close to the Ca2+ channels renders them with maximal repolarizing effect.

immunohistochemistry; ultrastructure; KVLQT1; human ether-a-go-go-related gene; long QT syndrome

K+ CHANNELS CONSTITUTE the largest, most diverse group of ion channels. In the heart, K+ channels play an essential role in the regulation of the cardiac action potential. Initial repolarization of the cardiac action potential is mediated by several depolarization-activated transient outward rectifier K+ channels, followed by the opening of the slowly activated delayed rectifier K+ channels, which terminate the repolarization step. In most cardiac cells, multiple components of the delayed rectifier K+ current (IK) have been identified. At least three distinct components of IK, referred to as ultrarapid IK (IKur), rapid IK (IKr), and slow IK (IKs), can be distinguished on the basis of kinetics of activation and pharmacological properties (10, 23, 28, 35, 42, 43).

Considerable progress has been made in identifying the molecular correlates of the above-mentioned components of IK. In particular, two types of voltage-gated K+ (Kv) channels have gained attention in this respect. One type, called the ether-a-go-go-related gene (ERG) channel, is activated rapidly on depolarization and has thus been suggested to be responsible for the IKr current, whereas the other type, the slowly activating KCNQ-type K+ channels, has been proposed to be the channels involved in IKs (1, 3, 41). The ERG subfamily has three members (ERG1–3), but only one of these proteins, ERG1, is expressed in the heart (49, 57). Of the KCNQ family, five members (KCNQ1–5) have been identified this far and the presence of two of these (KCNQ1 and KCNQ4) in the heart has been demonstrated on the mRNA level (31, 55, 61). In addition, several regulatory {beta}-subunits, called KCNEs, have been shown to interact with the pore-forming ERG and KCNQ channels. The function of KCNQ1 can be modulated by all the accessory subunits KCNE1–5 (1–3, 25, 41, 45, 52) and KCNQ1+KCNE1 is thought to underlie IKs (3, 41). ERG1 has been shown to interact with KCNE2 (MiRP1), and this complex has been suggested to coassemble to form IKr channels (1), although this notion has recently been challenged by the fact that KCNE2 and human ERG1 (hERG1) expressed in mammalian cells do not recapitulate the native IKr current (58). In equine cardiac tissue, ERG1 coimmunoprecipitates with KCNE1 [minK (16)], stressing the complicated picture of interactions.

The cardiac myocyte plasma membrane is a complex structure containing an outer sarcolemma and the transverse-axial tubular systems (TATS). TATS are invaginations of the plasma membrane into the cell interior and shortens the diffusion distance and time required to transport molecules between the inside of the cell and the extracellular phase. Several molecules, including ion channels and exchangers involved in the cardiac action potential and the exitation-contraction coupling, have been localized to both the outer sarcolemma and the transverse tubules. The voltage-gated Na+ channel rH1 and several K+ channels [Kv4.2, inward rectifier K+ 2.1 (Kir2.1), and TASK-1] involved in the different steps of the repolarization phase of the cardiac action potential have been demonstrated to be enriched in the T tubules (9, 29, 46, 51), whereas other K+ channels (Kv1.2, Kv1.5, and Kv2.1) appear to be concentrated in the outer sarcolemma (4).

The subcellular localization of KCNQ1 and ERG1 in the heart is not clear and has not been investigated in detail. In 1997, Brahmajothi and colleagues (7) demonstrated the presence of ERG1 in ferret heart atrial and ventricular myocytes, but the subcellular localization of the channel was not determined. Two other studies (38, 40) have indicated expression of ERG1 in a T-tubular-like structure of rat ventricle. A recent study by Franco and co-workers (18) described the overall distribution of KCNQ1 and ERG1 in the developing mouse heart; however, the limited resolution of the microscopical methods used left the exact subcellular distribution of the channels undetermined.

Although mRNA for both KCNQ1 and ERG1 has been detected in the mouse and rat heart (13, 18, 27, 38, 59, 60) and ERG1 protein expression has been demonstrated in the heart from both species (38, 40), the expression of the two delayed rectifier channels in rat and mouse cardiomyocytes remains controversial because functional IKs and IKr appear to be absent or present at very low densities (13, 27, 38). It therefore remains an open question as to whether the two proteins are expressed functionally in rat and mouse cardiomyocytes and what the exact physiological functions of the channels are.

In the present study, we produced an antibody directed against the COOH terminus of the hERG1 channel. We examined the protein expression of ERG1 and KCNQ1 in rat heart by Western blot analysis and determined the subcellular localization of the two channels in adult rat atrial and ventricular myocytes by confocal immunocytochemistry and immunoelectron microscopy to determine the specific localization of each channel in cardiac muscle cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibody production. A polyclonal serum was raised against hERG1 protein (Genbank accession no. U69885 [GenBank] ). The serum was raised against residue position 1037–1055 using the sequence DVESRLLDALQRQLNRLE [anti-hERG1(1037–1055)]. The antibodies were raised as described in detail previously (30).

Antibodies. The primary antibodies used in this study included, in addition to the produced hERG1 antibody, a polyclonal rabbit anti-ERG1 antibody (Alomone Labs; Jerusalem, Israel), a polyclonal goat anti-KCNQ1 antibody (Santa Cruz Biotechnology), a polyclonal rabbit anti-KCNQ1 antibody (Q1C2, gift from T. Jentsch) (12), and a monoclonal mouse anti-dihydropyridine receptor (DHPR) {alpha}2-antibody (Affinity Bioreagents). The antibody specificity and the absence of cross-reactions were verified by omission of the primary antibody.

Immunoblot analysis. Purified adult rat cardiac membranes (40 µg/lane) and whole cell lysates of transiently transfected COS-1 cells were separated by 8% SDS-PAGE using the minigel system (Bio-Rad Laboratories; Hercules, CA). Proteins were transferred onto a hybond-P polyvinylidene difluoride transfer membrane (0.45 µm; Amersham Biosciences) in 25 mM Tris base, 200 mM glycine, and 20% methanol with the use of a mini transblot (Bio-Rad). After transfer, the membranes were incubated overnight at 4°C in blocking buffer (PBS containing 5% lowfat milk powder and 0.1% Tween 20). The membrane was incubated for 2 h at room temperature in blocking buffer containing primary antibody [affinity-purified anti-hERG1(1037–1055) (0.6 µg/ml specific IgG; batch 040102/300/fin), rabbit anti-ERG1 (1/250 dilution), goat anti-KCNQ1 antibody (1/250 dilution) or rabbit anti-KCNQ1 (Q1C2, 1/250 dilution)]. After membranes were washed, the bound antibody was revealed with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG [F(ab')2 fragment] and HRP-conjugated donkey anti-goat antibodies (1/10,000, Jackson Immunoresearch Laboratories; West Grove, PA) in blocking buffer for 30 min, followed by visualization with the ECLPlus detection system (Amersham Biosciences) according to the manufacturer's instructions. The immunoblots were exposed on Hyperfilm ECL (Amersham Biosciences).

Fluorescence immunocytochemistry. Four male adult Wistar rats were deeply anesthetized with tribromoethanol and intracardially perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). The heart was removed and postfixed overnight at 4°C in 4% PFA. The heart was then dehydrated and embedded in paraffin, and 5-µm-thick sections were cut. The sections were redehydrated in a toluene ethanol series and microwave oven heated in a 10 mmol/l citric acid buffer. The sections were washed in PBS and incubated for 30 min in blocking buffer (0.2% fish skin gelatin and 0.1% Triton X-100 in PBS). They were subsequently incubated with the primary antibody [goat anti-KCNQ1 antibody, rabbit, rabbit anti-hERG1(1037–1055) crude serum (sixth bleed), or mouse anti-DHPR {alpha}2 antibody, all at 1:100 dilutions] overnight at 4°C. The sections were washed and incubated with secondary biotinylated antibodies (biotinylated donkey anti-rabbit, donkey anti-goat, or donkey anti-mouse antibodies, 1:500; Jackson ImmunoResearch) for 2 h at room temperature. This was followed by an 45-min incubation with StreptAvidin-Biotin-Complex (Elite, Vector; Burlingame, CA). The sections were then exposed to biotinylated tyramide (TSA indirect; NEN Life Science Products) in PBS containing 0.0015% H2O2 for 10 min, followed by a 1-h incubation with Alexa fluor 488-coupled streptavidin or Alexa fluor 594-coupled streptavidin (1:200, Molecular Probes; Leiden, The Netherlands). The stained sections were subsequently mounted in Prolong antifade (Molecular Probes).

The animal care procedures used in this study followed the guidelines of the Danish National Committee for Animal Studies.

Double fluorescence immunocytochemistry. For double fluorescence immunocytochemistry, adult rat heart paraffin sections (see Fluorescence immunocytochemistry) were incubated overnight at 4°C in both primary antibodies simultaneously: goat anti-KCNQ1 antibody and rabbit anti-hERG1(1037–1055) crude serum (sixth bleed), both at 1:100 dilutions. Primary antibodies were detected with an Alexa fluor 568 donkey anti-goat antibody (1:800, Molecular Probes) and an Alexa fluor 488 donkey anti-rabbit antibody (1:200, Molecular Probes). Sections were mounted in Prolong antifade (Molecular Probes).

Confocal microscopy and imaging. Laser scanning confocal microscopy was performed with the use of the Leica TCS SP2 system equipped with argon and He-Ne lasers. The objective was x63 W, the numerical aperture was 1.2. For double-labeling experiments, sequential scanning was employed to allow separation of signals from the two channels. Images were treated with the use of MetaMorph imaging software (Universal Imaging; Downington, PA) and Adobe PhotoShop version 5.5.

Immunoelectron microscopy. For immunoelectron microscopy, adult Wistar rats were deeply anesthetized with tribromoethanol and intracardially perfused with 4% PFA-0.1% glutaraldehyde in PBS. The heart was removed and postfixed overnight at 4°C in 4% PFA-0.1% glutaraldehyde.

The visualization of KCNQ1 and ERG1 at the ultrastructural levels was performed by using a preembedding technique, as previously described (26): 100-µm-thick vibratome sections were cut and collected in PBS. The sections were then incubated in 1% NaBH4 and 0.1% NaIO4 in PBS for 30 min and washed in PBS. This was followed by cryoprotection in 25% sucrose in water for 1 h and freezing for 5 s in liquid nitrogen. After being washed in PBS, the sections were processed in the following manner: incubation in 5% porcine serum in PBS for 30 min, followed by incubation in the primary antibodies [rabbit anti-KCNQ1 diluted 1:100 or rabbit anti-ERG1(1037–1055) crude serum (sixth bleed) diluted 1:500] in PBS with 1% BSA for 24–48 h at 4°C. After being rinsed with PBS with 0.25% BSA, sections were incubated overnight in biotinylated swine anti-mouse IgG (no. E464, diluted 1:400 in 0.25% BSA in PBS; DAKO; Copenhagen, Denmark), washed, and then incubated in blocking buffer (DuPont NEN) for 20 min. The sections were then incubated in streptavidin biotin-HRP complexes (Vector) for 60 min at room temperature, washed, and incubated in biotinylated tyramide (DuPont NEN, diluted 1/50) for 60 min. After being washed and incubated a second time in streptavidin-biotin-HRP complexes for 60 min, sections were washed in PBS and then in Tris·HCl (pH 7.6) and then incubated in 0.05% 3,3'-diaminobenzidine-HCl with 0.01% H2O2 in Tris·HCl for 15 min.

The sections were then dehydrated in a series of ethanol (30%, 50%, 70%, and 96%), block stained in 1% uranyl acetate in absolute ethanol for 1 h, rinsed in 2x absolute ethanol, and embedded via propylene oxide in Epon.

Two-micrometer-thick survey sections of the ventricles and atria were cut and counterstained with toluidine blue. Ultrathin sections with a gray interference color were cut from preselected areas and poststained in uranyl acetate and lead citrate. The sections were viewed and photographed with an electron microscope (model 208, Philips).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of hERG1 antibody. To investigate the localization of ERG1 in rat heart tissue, we produced an antibody directed against an epitope of the COOH terminus of the hERG1 channel. The antibody was tested for specificity by immunocytochemistry and Western blot analysis on COS-1 and human embryonic kidney (HEK)-293 cells transiently transfected with cDNA constructs encoding hERG1 and KCNQ1. The constructs coexpress green fluorescent protein, which allowed the identification of the transfected cells. The immunohistochemical experiments revealed that COS-1 cells transfected with hERG1 cDNA were stained when probed with the antibody (Fig. 1A,b), whereas no staining was detected in KCNQ1-transfected cells (Fig. 1A,a). HERG1 immunostaining was brightest in the perinuclear region in consistence with earlier reports (15). By Western blot analysis, the antibody recognized two strongly immunoreactive bands of 135 and 160 kDa in extracts of HEK-293 cells transfected with hERG1 (Fig. 1C). The sizes of the bands correspond to the reported molecular masses of immature core-glycosylated ERG1 protein and fully glycosylated mature ERG1 protein, respectively (15, 40). In addition, a band of ~75 kDa was recognized. This band is attributed to background because it was recognized in control lysates.



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  		Fig. 1. Characterization of human ether-a-go-go-related gene-1 (hERG1) and KCNQ1 antibodies. A and B: COS-1 cells were transiently transfected with KCNQ1 (A,a and B,b) and ERG1 (A,b and B,a). The cells were probed with antibodies directed against hERG1 (A) and KCNQ1 (B), respectively. Scale bars = 20 µm. C and D: extracts of control (Cont; nontransfected) human embryonic kidney (HEK)-293 (C) or COS-1 cells (D), HEK-293 (C), or COS-1 cells (D) transiently transfected with hERG1, KCNQ1, and sKvLQT1. cDNA and adult rat cardiac membranes (40 µg of protein/lane) were separated by 8% SDS-PAGE under reducing conditions and immunoblotted using antibodies directed against hERG1 (C) or KCNQ1 (D) as indicated. See MATERIALS AND METHODS for further details.

 

As also shown in Fig. 1C, the antibody recognized an immunoreactive band of 165 kDa in Western blots of adult rat cardiac membranes in accordance with earlier reports (38). A band of similar size was recognized by a commercially available ERG1 antibody (Fig. 1C; APC-016). In addition, a weak band of ~100 kDa was observed with both antibodies; however, the band was present in some rat cardiac membrane preparations but not in others (see DISCUSSION). None of the two antibodies detected the high-molecular-mass band (>200 kDa) described by Pond and colleagues (38).

Because KCNQ1 has never been detected at the protein level in the adult rat heart, we performed Western blot analysis of rat cardiac membranes and probed with a commercially available goat anti-KCNQ1 antibody. As shown in Fig. 1D, a band of 66 kDa corresponding to the reported molecular mass of full-length KCNQ1 protein (12) was detected. Interestingly, a band of lower molecular mass (~54 kDa) was also detected. Similar results were obtained when probing with a rabbit anti-KCNQ1 antibody (Fig. 1D, Q1C2), the specificity of which has been described elsewhere (12). To ensure that the commercial antibody was specific, we also performed immunohistochemistry and Western blot analysis on COS-1 cells transiently transfected with cDNA constructs encoding KCNQ1 and hERG1. As shown, the antibody specifically recognized KCNQ1-transfected cells (Fig. 1B,b) but not hERG1-transfected cells (Fig. 1B,a) by immunocytochemistry. By Western blot analysis, the antibody specifically recognized a band of 66 kDa in extracts of COS-1 cells transfected with KCNQ1 but not in extracts of control COS-1 cells or cells transfected with hERG1 (Fig. 1D). We furthermore examined the KCNQ1 variant cloned by Sanguinetti and co-workers (41) and later named sKvLQT1 (44). The sKvLQT1 protein is 95 residues shorter than wild-type KCNQ1 and, as expected, the mobility of this protein was shifted to a lower molecular mass (~50 kDa) compared with wild type (Fig. 1D).

Immunohistochemical localization of KCNQ1 and ERG1 in rat heart. The subcellular localization of KCNQ1 and ERG1 in adult rat heart was examined using the commercial goat anti-KCNQ1 and the produced hERG1 antibodies. The localization was carried out with the use of tyramide-amplified immunofluorescence analysis of rat heart tissue sections. Tyramide amplification was chosen to obtain strong fluorescence signals to get a clear overview of the localization of the two channels.

Confocal microscopy of adult rat atrial tissue revealed prominent staining for KCNQ1 and some staining for ERG1 of the peripheral sarcolemma (Fig. 2, A and B). Surprisingly, a prominent transverse-oriented striated fluorescence labeling for both ion channels was also observed. The transverse bands were separated by ~2 µm, which would indicate T-tubular localization. This was somewhat unexpected in atrial cells because they are thought to be essentially devoid of T tubules (17). To examine this further, we looked at the distribution of the sarcolemmal DHPR {alpha}2-subunit in atrial cells. DHPR is a voltage-dependent L-type Ca2+ channel, which forms part of the excitation-contraction coupling apparatus in cardiac myocytes. The channel has been localized to coupling sites (dyads) in the peripheral sarcolemma and in T tubules in ventricular tissue (8). In contrast to earlier studies, which limited DHPR distribution to the peripheral sarcolemma in rabbit atrial cells (8), we found the protein to be expressed in both the peripheral sarcolemma and in the cytoplasm as regularly spaced transverse bands (Fig. 2C). The distribution correlated well with that observed for KCNQ1 and ERG1.



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  		Fig. 2. Confocal images of adult rat atrial (A–C) and ventricular (D–F) tissue sections immunolabeled for KCNQ1 (A and D), ERG1 (B and E), and the dihydropyridine receptor (DHPR) {alpha}2-subunit (C and F). A2–F2 show enlargements of the regions indicated in A1–F1, respectively. Labeling of both potassium channels was observed in the peripheral sarcolemma (arrow) as well as in transverse oriented stripes (arrowhead) of atrial cells (A and B). Note how the labeling resembles that of DHPR (C). In ventricular tissue, both KCNQ1 (D) and ERG1 (E) were observed in transverse-oriented stripes (arrowheads), whereas KCNQ1 was also observed in the peripheral sarcolemma (arrow in D2). ERG1 labeling in the peripheral sarcolemma was restricted to the mouth of the T tubules (arrow in E2). The bar represents 20 µm.

 

In adult rat ventricular tissue, ERG1 channels were primarily observed as regularly spaced transverse bands with a periodicity of ~2 µm (Fig. 2E). Similar to what had been observed in atrial tissue, the ERG1 channels were expressed at low levels in the peripheral sarcolemma. KCNQ1, however, was detected as patches in the sarcolemma to a greater extent than ERG1. Labeling for KCNQ1 was observed in the peripheral sarcolemma as well as in what appeared to be the T tubules (Fig. 2D). In comparison, the DHPR {alpha}2-subunit localization resembled the ERG1 labeling (Fig. 2F). Because DHPR has been localized to the T-tubular system system of rat and rabbit ventricular myocytes (8, 21, 46, 50, 62), this observation indicates that both KCNQ1 and ERG1 are located in the T-tubular system, whereas KCNQ1, in addition, is also expressed in the peripheral sarcolemma.

Ultrastructural immunolocalization of KCNQ1 and ERG1 in rat heart. To study the subcellular localization of KCNQ1 and ERG1 in rat myocytes with higher resolution, we performed electron microscopy analysis of adult rat atrial and ventricular tissue sections labeled for each ion channel. In these experiments, the rabbit anti-KCNQ1 and the produced rabbit anti-hERG1 antibodies were employed.

In the atria, the immunoreactivity for both ERG1 and KCNQ1 was observed along the Z lines (Fig. 3). In the areas between the myofibrils, the immunoreactivity was confined to tubular structures of the atrial myocytes. In the sections immunoreacted for KCNQ1, the reaction product was also observed along the plasmalemma of the myocytes (Fig. 3, B and D).



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  		Fig. 3. Electron micrographs from sections of the left adult rat atrium. A and C: tissue has been reacted with antibodies against hERG1. The immunoreaction is located above the Z lines (arrows in A). A stained transverse tubular structure is seen in C (arrow). The cell membrane of the myocytes was nearly devoid of staining. B and D: tissue has been reacted with antibodies against KCNQ1. Staining is observed corresponding to the Z lines, but a moderate staining of the cell membrane of the myocytes is also observed (arrowheads). Bars = 2 µm (A), 1 µm (B), and 0.5 µm (C and D).

 

In the ventricles, survey electron micrographs stained with antibodies against ERG1 showed a prominent labeling of the T-tubular system (Fig. 4A). At higher magnification, the contact area with the smooth endoplasmic reticulum, the dyad, was observed (Fig. 4, B and C). The reaction product was in the dyads confined to the T tubules, whereas the endoplasmic reticulum remained unstained (Fig. 4, B and C). The labeling in the peripheral sarcolemma was sparse and concentrated at the mouth of the T tubules (Fig. 4D). In contrast, strong signals were observed in the T tubules. ERG1 appeared to be concentrated in this structure.



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  		Fig. 4. Electron micrographs of cardiac muscle cells of the left ventricle of the adult rat immunoreacted for the ERG1 channel protein. A strong immunoreactivity is seen in the T-tubular system (arrows in A) and in the cell membrane (arrows, in D) close to the openings of the T tubules. In B and C, the dyads (arrows) are observed and only the T tubulus is stained. Scale bars = 2 µm in A and 1 µm in B–D.

 

When antibodies against KCNQ1 were used, a strong labeling of the sarcolemma was observed (Fig. 5, A and B). The labeling exhibited a punctate distribution (Fig. 5B). The T-tubular system was also labeled, but immunoreactivity was generally less dense than the immunoreactivity confined to the cell membrane (Fig. 5A).



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  		Fig. 5. Electron micrographs of cardiac muscle cells of the left ventricle of the adult rat immunoreacted for the KCNQ1 channel protein. The cell membrane is strongly immunoreactive [arrows in A, but also the T-tubular system is stained (arrowheads in A)]. The strong immunoreactivity in the cell membrane exhibits a patchy distribution of the cytoplasmic site of the cell membrane (arrows in B). Scale bars = 1 µm.

 

Differential distribution of KCNQ1 and ERG1 in rat ventricles. To examine the subcellular localizations of KCNQ1 and ERG1 relative to each other, colabeling experiments were carried out. In these experiments, no tyramide amplification was employed. Tyramide amplification makes use of the enzymatic action of HRP and therefore involves a greater risk of diffusion of reaction product. For that reason, classic immunocytochemistry was employed to obtain a more accurate picture of channel localization.

As demonstrated in Fig. 6, both ERG1 and KCNQ1 displayed well-ordered arrays of fluorescent spots, indicating that the channels were distributed in discrete clusters. Note also that KCNQ1 labeling is associated with the peripheral sarcolemma to a far greater extent than ERG1, which appears restricted to the T tubules. In the T tubules, KCNQ1 and ERG1 immunolabeling was often observed closely associated, but they only colocalized to a small extent.



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  		Fig. 6. Confocal scanning images of adult rat ventricular tissue double labeled for KCNQ1 (A) and ERG1 (B). Both channels appeared as discrete clusters in the cell membrane. C shows an overlay of images A and B. Although often expressed in the proximity of each other, the KCNQ1 (arrowhead) and ERG1 (arrow) labelings did not colocalize (C). A2–C2 show an enlargement of the region indicated in C1. Scale bar 10 µm.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Subcellar localization of ERG1 and KCNQ1. In the present study, we have demonstrated the expression of both KCNQ1 and ERG1 in the adult rat heart and examined the subcellular distribution of the two channels. To our knowledge, this is the first study reporting the presence of KCNQ1 channels in the rat heart at the protein level. The data are strongly supported by the fact that two different antibodies directed against KCNQ1 were employed, and, furthermore, Western blot analysis, immunohistochemistry, and electron microscopical methods were all used to demonstrate the presence of the protein. The very robust KCNQ1 protein expression was quite surprising given that in adult mice and rat IKs is present only at low densities, possible due to downregulation of KCNE1 during maturation (13, 27). However, because KCNQ1 is capable of interaction with all other members of the KCNE family, it can be speculated that KCNQ1 contributes to other K+ currents than IKs in rat myocytes. In support of our data, a role for KCNQ1 in the mouse heart has recently been suggested in relation to sympathetic stimulation (53). In KCNQ1-targeted deletion mice, long QT phenotypes were observed in response to sympathetic stimulation indicating a role for KCNQ1 in cardiac repolarization in the presence of catecholamines.

Interestingly, by Western blot analysis, two bands, 66 and 54 kDa, respectively, were detected by both KCNQ1 antibodies in the rat heart. This observation suggests that truncated KCNQ1 proteins are expressed in the rat heart. In support of this observation, a truncated splice variant of KCNQ1 has been cloned from the rat heart (60). When expressed along with full-length KCNQ1, this truncated version of the channel suppressed the KCNQ1 current. It can thus be speculated that dominant negative current suppression by a truncated form of KCNQ1 underlies the lack of functional Iks currents measurable in adult rat cardiomyocytes. The lower-molecular-mass band (54 kDa) was not detected in human or guinea pig heart membranes (data not shown).

In Western blots of rat cardiac membranes probed with rabbit anti-hERG1(1037–1055) antibody a band of 165 kDa was detected corresponding to the reported molecular mass of full-length rat ERG1 (rERG1) (38). However, in contrast to an earlier report, our antibody did not detect the high-molecularmass form (205 kDa) of ERG1 reported to be expressed in rat and mouse hearts (38). A possible explanation for this discrepancy could be the use of different antibodies because only one of two antibodies produced in the above mentioned study recognized the rat cardiac 205-kDa version of ERG1. In addition, a commercial ERG1 antibody failed to detect the higher-molecular-mass form in our study. Both the APC-016 and rabbit anti-hERG1(1037–1055) antibodies detected a very weak band of 100 kDa in rat heart membranes. The identity of this band is unknown. An NH2 terminal splice variant of ERG1 with similar molecular mass has been cloned from mouse (mERG1b) and human (hERG1b) hearts (33, 36); however, we do not know whether the 100-kDa band observed in our study corresponds to rERG1b. A band of similar size has been detected in the equine heart (16) and was detected in rat brain in one study (37) but was not observed in another (38). Again, a possible explanation for the different observations could be the use of different antibodies or the fact that this band is weak and observed only in some preparations.

In adult rat atrial tissue, KCNQ1 was strongly expressed in the peripheral sarcolemma. Furthermore, both ion channels were observed in transverse bands separated by ~2 µm, which would indicate T-tubular localization. A similar distribution pattern was observed for the DHPR {alpha}2-subunit. The T tubules are rare in rat atrial myocytes; however, rat atrial cells exhibiting a highly developed network of T-tubular structures have been described (17). In rat atrial cells, the T tubules are predominantly oriented parallel to the myofibrils along the longitudinal axis of the muscle fiber, although transverse structures are also observed (17). In our study, the immunoreactivity along the Z lines was confined to transverse tubular structures. In addition, immunoreactivity was also seen in tubular structures, close to the transverse tubuli, but with their axis in a longitudinal direction, indicating T-tubular localization.

In the adult rat ventricle, ERG1 was primarily located in the T tubules, whereas KCNQ1 was found in both the T tubules and the peripheral sarcolemma. A T-tubular localization of the channels is not unexpected because several ion channels and transporters involved in the excitation-contraction process recently have been localized to these sites in different species. This includes, among others, the K+ channels Kv4.2 (51), Kir2.1 (9), TASK-1 (29), the voltage-gated Na+ channel rH1 (11, 46), the {alpha}1-, {alpha}2-, and {beta}2-subunits of DHPR (8, 9, 21, 46), and the cardiac Na+/Ca2+ exchanger (19, 46). Furthermore, ERG1 has already by immunohistochemistry been localized to a T-tubular-like structure of rat ventricles (38, 40) and the KCNQ1-associated protein minK has been shown to interact with a T-tubular protein (20). The T-tubular localization of the channels allows fast spreading of the repolarization and locates KCNQ1 and ERG1 in the proximity of other key regulators of exitation-contraction coupling.

ERG1 has been reported to be expressed in both the sarcolemma and T tubules of the rat ventricle depending on the antibody used (38). However, we observed very little sarcolemmal labeling and primarily at the mouth of the T tubules. The reason for this discrepancy remains unclear, but it could be because our antibody does not recognize the high-molecularmass form (~205 kDa) of ERG1, which has been suggested to be targeted to the sarcolemma (38). In support of this explanation, only one of two ERG1 antibodies produced in the above-mentioned study recognized the high-molecular-mass form of ERG1 from the rat heart and only one of the two antibodies labeled the peripheral sarcolemma (38).

Electron microscopy furthermore revealed strong expression of KCNQ1 in the peripheral sarcolemma. Because the T tubule is the membrane structure predominating in excitation-contraction coupling (5, 6, 14, 39, 47, 50, 56, 62), it is fascinating to speculate that the role of the sarcolemmal KCNQ1 channels is distinct from the T-tubular KCNQ1 channels. Whereas the most obvious function of T-tubular KCNQ1 channels is to counteract and overrule the depolarizing action of the Ca2+ currents (DHPR) during the repolarization of the cardiac action potential, the sarcolemmal KCNQ1 channels could play another role. Our group and others (24, 32) have recently shown that KCNQ1 channels are activated by cell swelling and volume regulation could thus be the primary function of the sarcolemmal KCNQ1 channels.

Discrete localization of KCNQ1 and ERG1 channel clusters in sarcolemma/T tubule system. The present study revealed that the KCNQ1 and ERG1 channels could be detected as discretely localized clusters in the cell membrane. In neurons, ion channels have been reported to be localized in the plasma membrane through binding to intracellular proteins (48, 54) and in cardiac myocytes both the Na+/Ca2+ exchangers and Na+ channels bind to cytoskeletal proteins (22, 34). It is therefore likely that the highly ordered localization patterns of KCNQ1 and ERG1 reflect the interaction of the channels with intracellular proteins that are essential for the localization of the channels. In favor of this hypothesis, a recent study by Furakawa and colleagues (20) demonstrated the interaction, in the rat, of the KCNQ1 accessory {beta}-subunit minK with the sarcomeric protein T-cap, which would localize the KCNQ1/minK channel complex to the T-tubular membrane. The slightly differential subcellular distributions of KCNQ1 and ERG1 channels in the T tubules could reflect that different proteins are responsible for the clustering and localization of the two channels. No anchoring proteins responsible for the subcellular localization of KCNQ1 and ERG1 channels have been identified yet, and identification of such proteins is therefore of great interest.


   ACKNOWLEDGMENTS
 
We thank T. Jentsch (Universität Hamburg, Germany) for supplying the antibody against KCNQ1 (rabbit anti-KCNQ1) and Ursula Rentzman for excellent technical assistance.

GRANTS

This work was supported by Danish Heart Foundation Grant 01-1-2-51-22905A, The Danish Medical and Natural Sciences Research Councils, The Novo Nordisk Foundation, The John and Birthe Meyer Foundation, and The Velux Foundation. H. B. Rasmussen was supported by Danish Heart Foundation Grants 00-2-38A-22850 and 01-1-2-51-22905A/B.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. B. Rasmussen, Univ. of Copenhagen, Dept. of Medical Physiology, The Panum Institute, Bldg. 12.5.10, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark (E-mail: hannebr{at}mfi.ku.dk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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