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Cardiovascular Research 2002 56(1):93-103; doi:10.1016/S0008-6363(02)00498-4
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Copyright © 2002, European Society of Cardiology

Cardiac-enriched LIM domain protein fhl2 is required to generate IKs in a heterologous system

Sabina Kupershmidta,*, Iris C.-H. Yangb, Margaret Sutherlanda,1, K.Sam Wellsc, Tao Yanga, Ping Yanga, Jeffrey R. Balsera,b and Dan M. Rodena,d

aDepartment of Pharmacology, 561A PRB, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, 532C Robinson Research Building, 23rd Avenue South at Pierce Avenue, Nashville, TN 37232-6602, USA
bDepartment of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA
cDepartment of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA
dDepartment of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA

* Corresponding author. Tel.: +1-615-936-2586; fax: +1-615-322-4707 sabina.kupershmidt{at}vanderbilt.edu

Received 12 December 2001; accepted 17 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Co-expression of the KvLQT1 and minK potassium channel subunits is required to recapitulate IKs, the slow component of the cardiac delayed rectifier current, and mutations in either gene cause the congenital Long QT syndrome. It is becoming increasingly well-recognized that multiprotein channel complexes containing proteins capable of modulating channel function assemble at the plasma membrane. Thus, the aim of our study was to identify proteins involved in IKs modulation. Methods and results: Using a yeast-two-hybrid screen with the intracytoplasmic C-terminus of minK as bait, we identified the cardiac-enriched four-and-a-half LIM domain-containing protein (fhl2) as a potential minK partner. We show interaction between the two proteins in GST pulldown assays and demonstrate overlapping subcellular localization using immunocytochemistry of transfected cells supporting a potential interaction. At the functional level, expression of KvLQT1and minK in HEK cells, which lack endogenous fhl2 protein, generated IKs only when fhl2 was co-expressed. By contrast, in CHO-K1 cells, which express fhl2 endogenously, IKs was suppressed by anti-fhl2 antisense which did not affect the currents generated by KvLQT1alone. Conclusion: These data indicate that at least in heterologous cells, the generation of IKs requires fhl2 as an additional protein component.

KEYWORDS Arrhythmia (mechanisms); Ion channels; K-channel; Long QT syndrome; Ventricular arrhythmias


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Voltage-gated potassium channels comprise a large family of membrane proteins that regulate the membrane potential of excitable cells in the heart, central nervous system, and other organs. Functional potassium channel diversity originates in part by the expression of a large number of different, pore-forming {alpha} subunit proteins, and in part by the interaction of {alpha} subunit multimers with β subunits (reviewed in Ref. [1]). The delayed rectifier IKs, which plays an important role during repolarization of the cardiac action potential, illustrates this paradigm: co-expression of the minK (KCNE1) and KvLQT1 (KCNQ1) gene products is required to recapitulate IKs in heterologous systems [2,3]. KvLQT1 acts as the {alpha} subunit and can generate a current (activating much more rapidly than IKs) when expressed by itself [4]. MinK is a small protein with a single transmembrane domain and no classical pore region. It does not generate a current autonomously, but functions to increase the amplitude of the KvLQT1-generated currents and to alter gating [2–4], unitary current [5,6] and pharmacological properties [7,8] to recapitulate IKs. Loss of function mutations in either KCNQ1 or KCNE1 produce defective repolarization, syncope, and sudden death due to arrhythmias in the congenital Long QT syndrome [9].

It is well established that potassium channels are associated with modulating proteins that are not directly involved in pore formation, but serve other functions such as signaling [10,11] or anchoring to specific loci within the cell [12–14]. In order to refine the molecular basis of IKs physiology, we used the minK intracytoplasmic C-terminus as ‘bait’ in a yeast-two hybrid screen, and evaluated the functional consequences of a potential minK partner in cultured cells. Our studies demonstrate that the cardiac-enriched protein fhl2 (named after its ‘four and a half LIM’ domains) [15] interacts with minK and is required to recapitulate IKs. The LIM structural motif is composed of two adjacent zinc fingers that are involved in protein–protein interactions [16]. Proteins in this family may also contain functional domains such as homeodomains [17], PDZ [18], or kinase domains [19], but others, including fhl2, consist of LIM domains only. Several LIM proteins associate with cytoskeletal elements [20–22] and in human cardiac myocytes fhl2 has been shown to co-localize with {alpha}-actinin at the Z-lines [23] where minK has recently also been demonstrated [24]. The present study identifies fhl2 an additional player in the IKs complex and will allow us to further define its role in cardiac physiology.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Yeast two hybrid screening
To generate a bait plasmid, we cloned the 63 C-terminal amino acids of minK into the vector pGBDU-C2 [25], which carries a URA3 selection marker generating an in frame fusion with the GAL4 DNA binding domain (BD) of this vector. The resulting clone, MinK C-term, was sequenced to verify orientation of the insert and its correct reading frame. MinK C-term was transformed into yeast strain PJ69-4A (a kind gift from Dr P. James, University of Wisconsin Medical School) and grown on Synthetic Complete Dropout media lacking Uracil (SD/-ura). A liquid culture was transformed with a mouse brain Matchmaker cDNA library (Clontech, Palo Alto, CA) contained on the pACT2 vector. Yeast transformation was accomplished using the method of Gietz and Schiestl [26]. A total of 9.6x106 doubly transformed colonies were plated, as assayed on SD/-ura/-leu (leucine). The experimental transformation was initially plated on SD/-ura/-leu/-his (histidine) plus 2 mmol/l 3-amino-1,2,4-triazole. After 9 days of incubation at 30 °C, colonies were replica plated onto SD/-ura/-leu/-ade (adenine). White colonies growing on this selection were then re-grown on SD/-ura/-leu/-his for liquid β-gal assays according to recommendations by the manufacturer of the library (Clontech, Palo Alto, CA). KvLQT1 C-term was constructed by digesting a human KvLQT1 cDNA with BglII and BamHI. The resulting 1 kb fragment (encompassing amino acids 395–676) was then subcloned into the BglII site of pGBDU-C2. The fhl2 and HERG C-term prey vectors were constructed using PCR amplification from cDNA templates with primers incorporating EcoRI and BamHI linkers at their 5' and 3' ends, respectively. The resulting PCR fragments were ligated into EcoRI and BamHI cut pGAD-C2. The full-length fhl2 bait clone includes amino acids 2–279. LIM domains 1/2–2 includes amino acids 2 to 176, and LIM domains 3–4 includes amino acids 172–279. HERG C-term includes amino acids 1018–1159 of HERG [27].

2.2 Sequencing of inserts
Candidate yeast colonies were lysed in 10 µl of yeast lysis buffer (1.2 mol/l sorbitol, 100 mmol/l sodium phosphate, pH 7.4, and 2.5 mg/ml zymolyase (ICN, Canada)) for 10 min at 37 °C. Then 2 µl of the lysate was used as a template in a PCR reaction with pACT2-specific primers flanking the cDNA inserts. PCR fragments were sequenced using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Cleveland, OH).

2.3 RNA analysis
Northern hybridization was performed on total cell RNA as described previously [28] using a full-length fhl2 cDNA probe.

2.4 Constructs
The GST-minK fusion plasmid was constructed by ligating a PCR fragment coding for the last 63 amino acids of mouse minK in frame with the GST open reading frame into the EcoRI site of vector pGEX-2TK (Pharmacia, Amersham). EcoRI linkers and a stop codon were incorporated into the PCR primers. The entire fhl2 coding region was subcloned into pBKCMV (Stratagene, La Jolla, CA) following PCR amplification. Restriction enzyme sites as well as a stop codon were incorporated into the PCR primers. For sense- and antisense-fhl2 expression experiments, the entire fhl2 coding region was inserted in both orientations into the EcoRI site of vector GFPIre (a kind gift of Dr David Johns, Johns Hopkins School of Medicine) placing it downstream of the CMV promoter, the enhanced green fluorescent protein (EGFP) cistron, and a poliovirus internal ribosome entry site (IRE).

2.5 Cell culture and transfections
Chinese hamster ovary K1 (CHO-K1) cells were cultured in Ham’s F-12 media and HEK 293 and tsA201 cells in DMEM. Cells were transiently transfected using the lipofectamine transfection reagents and method (Gibco-BRL). A GFP expressing vector was co-transfected and cells displaying green fluorescence 48–72 h after transfection were used for electrophysiologic experiments.

2.6 Preparation of protein extracts
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Mouse hearts were harvested and minced in lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mmol/l NaCl, 50 mmol/l Tris, 1 mmol/l EDTA). The homogenates were then passed through a Centricon Plus-20 column (Millipore, Bedford, MA).

2.7 GST pulldown assays
A GST–minK fusion plasmid containing the C-terminal 63 amino acids of minK in vector pGEX-2TK was transformed into BL-21 E. coli. Expression and purification of the fusion protein was carried out according to the manufacturer’s recommendations (Amersham Pharmacia, Biotech) and linked to glutathione Sepharose 4B beads. Total mouse heart protein extracts were incubated with bacterially expressed GST–minK or GST alone for 1 h at RT. The complexes were then washed three times in RIPA buffer (0.1% SDS, 0.5% sodium deoxycholate and 0.5% NP-40 in PBS), boiled for 5 min in SDS gel loading buffer and run on a 10% SDS PAGE for Western analysis.

2.8 Western blotting
Membranes were blocked overnight in TS (50 mmol/l Tris, 150 mmol/l NaCl) plus 10% goat serum (S1) at 4 °C. They were then incubated in primary antibody (1:500 for anti-fhl2) in TS plus 5% goat serum plus 0.05% Tween 20 (S2) and washed two times in S3 (50 mmol/l Tris, 0.5 mol/l NaCl, 5% goat serum, 0.05% Tween 20) followed by incubation with secondary donkey anti-rabbit horseradish peroxidase (HRP)-linked antibody (1:10 000, Amersham Pharmacia Biotech), in S2. This was followed by three washes in S1, S2, S3. The blots were developed using the ECL system (Amersham Pharmacia Biotech).

2.9 Immunocytochemistry
Transfected HEK 293 cells grown on 35-mm Mat-Tek dishes were fixed in 4% paraformaldehyde/PBS, washed, and preincubated in 5% goat serum, 2% BSA and 0.1% Triton X-100. This was followed by incubation with the anti-FLAG M2 (1:200, Sigma) and anti-fhl2 (1:50) primary antibody in 2% BSA/PBS overnight. Cells were then incubated with cy5-conjugated goat anti-mouse IgG and cy3-conjugated goat anti-rabbit IgG (1:400, Jackson Immuno Research) secondary antibody, respectively. The cells were imaged on a Zeiss LSM410 confocal imaging system using a laser for the excitation of cy3 (567 nm) and cy5 (647 nm).

2.10 Antibody production
A fhl2 immunogenic peptide was generated, linked to a KLH carrier, and rabbits were immunized (Quality Controlled Biochemicals, Hopkinton, MA). The peptide used was Ac-CGSSLVDKPFAAKEEQL-amide. Reactive antisera were immunoaffinity purified using a thiol coupling gel.

2.11 Patch-clamp analysis
Potassium currents were recorded at room temperature (RT, 20–22 °C) using the whole cell patch clamp technique. Electrode resistances were 1–2 M{Omega} when filled with a pipette intracellular solution containing: 110 mmol/l KCl; 5 mmol/l K2ATP, 2 mmol/l MgCl2; 10 mmol/l Hepes; and 5 mmol/l K4BAPTA, pH 7.2. The bath solution for all experiments contained: 145 mmol/l NaCl; 4 mmol/l KCl; 1.8 mmol/l CaCl2; 1.0 mmol/l MgCl2; 10 mmol/l HEPES; and 10 mmol/l glucose, pH 7.35. IKs or IKvLQT1 were activated from a holding potential of –80 mV with 4- or 1-s depolarizations, respectively, to voltages ranging from –60 to +60 mV in 20-mV increments. The membrane potential was then stepped to –50 mV and tail currents recorded. Patch clamp data were acquired using pCLAMP6 software (v6.0.4; Axon Instruments, Foster City, CA). Currents were filtered at 5 kHz (–3 dB, four-pole Bessel filter) and recorded using an Axopatch 200 integrating patch clamp amplifier (Axon Instruments) with 80% series resistance compensation. Pooled data are presented as means and standard errors, and statistical comparisons were made by Student’s t-test with P<0.05 considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Identification of fhl2 as a potential minK partner
Using the 63 intracytoplasmic C-terminal amino acids of minK as bait in a yeast two-hybrid screen of a mouse brain cDNA library, we isolated the four-and-a-half LIM domain protein—fhl2. The interacting clone included the sequence of all but the 25 N-terminal amino acids of fhl2. It reacted positive in all three selection systems: positive growth on SD/-ura/-leu/-his (Fig. 1), positive growth and generation of white colonies on SD/ura/-leu/-ade and a positive reaction in liquid β-gal assays.


Figure 1
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  		Fig. 1 Interactions of the minK C-terminus with full-length fhl2 in yeast. The bait constructs were transformed into yeast and grown on SD/-ura. Resulting colonies were grown in liquid culture and then supertransformed with the prey constructs and plated on SD/-ura/-leu and SD/-ura/-leu/-his. Only those plasmid combinations expressing proteins capable of interaction (minK C-term and full-length fhl2) activate the His reporter gene and can grow on His-deficient media.

 
The complete 1087 nucleotide cDNA, including the 829-nucleotide open reading frame (279 amino acids), was then amplified from mouse heart cDNA. Full-length fhl2 was required in conjunction with the minK C-terminus to activate the His reporter gene in yeast, whereas LIM domains 1/2 to 2 or LIM domains 3 and 4 by themselves were unable to do so (Fig. 1). This indicates that LIM domains 1–4 are required for the interaction with the minK C-terminus or that the junction between LIM domains 2 and 3 must be intact. Similarly, the fhl2 prey construct or the minK bait constructs were not able to interact with a KvLQT1 bait construct and a HERG (another K+ channel {alpha} subunit) [27] prey construct, respectively, indicating that the observed interaction is specific for minK and fhl2.

Fhl2 expression is known to be enriched in the heart [29] and Northern blotting revealed abundant fhl2 mRNA in heart. With prolonged exposure, expression could be detected in uterus and kidney, where minK is also expressed [30,31], as well as in brain, where our unpublished data indicate minK expression (Fig. 2).


Figure 2
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  		Fig. 2 Expression of minK and fhl2 in the adult murine CNS and heart. (A) Northern blot using total RNA from various mouse tissues and the fhl2 cDNA probe. The same blot was stripped and re-probed using a rat cyclophilin cDNA (lower portion) to allow for comparison of loading. RNA from brain is present in relatively greater amounts. (B) Northern blot analysis using the fhl2 cDNA probe on 20 µg of total RNA isolated from commonly used laboratory cell lines demonstrates that fhl2 RNA is widely expressed.

 
To test for an association of minK with fhl2, we generated a peptide-specific antibody against fhl2. This antibody detects a single band of the expected molecular weight (32 kDa) in protein extracts derived from mouse heart (Fig. 3A, lane 3). We tested the total mouse heart extract in a glutathione S-transferase (GST) pulldown assay with a bacterially expressed minK–GST protein fusion. A protein co-migrating with fhl2 from mouse heart extracts associated with GST–minK-linked beads (Fig. 3A, lane 1), whereas it did not associate with GST-linked beads alone (Fig. 3A, lane 2). FLAG-tagged minK and fhl2 also showed overlap of subcellular localization when assayed by confocal microscopy of HEK 293 cells transfected with the two cDNAs (Fig. 3B). Neither minK nor fhl2 were distributed uniformly, but rather were preferentially expressed within discrete foci. Despite this heterogeneous intracellular distribution and a relatively broader expression pattern of fhl2, areas of overlap (yellow regions) were detected and minK staining was generally not observed in areas where fhl2 expression was absent.


Figure 3
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  		Fig. 3 In vitro interactions of fhl2 and minK. (A) GST pulldown of fhl2 and minK. Western blot analysis using an anti-fhl2 antibody. The minK 63 C-terminal amino acids were fused in frame with the GST protein. Total protein extract from mouse heart was incubated with (1) the bead-linked GST–minK fusion protein, and (2) GST alone (unlinked to minK). (3) Mouse heart protein extract to indicate the position of fhl2. (B) Microscopic analysis of HEK 293 cells transfected with KvLQT1, fhl2 and FLAG2–minK. Magenta coloring (Cy5 fluorescence) indicates minK-specific labelling, and green coloring (Cy3 fluorescence) indicates fhl2-specific labeling. Coincident labeling appears yellow (some of it indicated by arrows). All images were acquired using a 40x/1.3 plan Neofluar objective on a Zeiss LSM410 confocal microscope.

 
3.2 Functional interactions of fhl2 and minK
Northern blot analysis readily identified fhl2 mRNA transcripts in five commonly used laboratory cell lines (COS, Ltk–, CHO-K1, HeLa, HEK 293) and in AT-1 cells (cardiac cells derived from a murine atrial tumor [32], Fig. 2B). However, we determined that HEK293 cells, in which fhl2 mRNA was least abundant (Fig. 2B), produced undetectable amounts of fhl2 protein, whereas protein was easily detected in CHO cells (Fig. 4A). HEK cells contain an endogenous outward current that can be readily distinguished from IKs because of its rapid activation and inactivation kinetics [33]. However, within our experimental paradigm, this background current can not be distinguished from the current evoked by KvLQT1 alone. When HEK293 cells were co-transfected with KvLQT1, minK and a third plasmid expressing the green fluorescent protein (GFP), IKs was not detected in 9/10 fluorescent cells. We detected a small tail current in a single transfected HEK cell and surmise that it may have been caused by the expression of KvLQT1 alone (Fig. 4B). The other nine transfected HEK cells showed outward current identical to the one shown in Fig. 4B, but no tail currents. By contrast, IKs was recorded in 9/13 transfected with KvLQT1, minK, and a bicistronic vector expressing GFP and fhl2 (Fig. 4C, P=0.06 for 1/10 vs. 9/13, or P=0.04 for 1/11 vs. 9/13 cells (chi-square analysis)).


Figure 4
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  		Fig. 4 The generation of IKs requires fhl2 expression. (A) Western blot analysis of total cell extracts using the anti-fhl2 polyclonal antibody. (B) Current tracing from HEK 293 cell transfected with minK and KvLQT1. (C) IKs recorded from HEK 293 cells transfected with fhl2 (sense construct) and KvLQT1 plus minK.

 
When the same set of experiments was performed in CHO-K1 cells, typical IKs was recorded from cells transfected with minK and KvLQT1 alone (Fig. 5A), and was indistinguishable from that recorded from cells transfected with minK, KvLQT1 and the fhl2 sense construct (Fig. 5C, circles vs. squares). By contrast, IKs amplitude was reduced by ~35% (P<0.05) in cells transfected with minK, KvLQT1 and an anti-fhl2 antisense construct (Fig. 5B and C, filled triangles). Neither sense nor antisense cDNA constructs altered IKs voltage dependency of activation (Table 1).


Figure 5
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  		Fig. 5 Antisense-fhl2 treatment suppresses whole-cell IKs magnitude. Currents recorded in CHO-K1 cells coexpressing KvLQT1 and minK are shown without (A) and with (B) cotransfection of anti-fhl2 antisense. (C) Summary data showing the current–voltage relationship of KvLQT1 plus minK currents. Anti-fhl2 antisense caused voltage-independent suppression of the KvLQT1+minK current magnitude (* P<0.05), but did not alter IKs activation or deactivation (see Table 1). The voltage clamp protocol is shown in the inset. Currents recorded from cells expressing KvLQT1 alone are shown without (D) and with (E) cotransfection of the antisense fhl2. Antisense had no effect on the deactivation kinetics or the current magnitude. (F) Current–voltage relationship of KvLQT1 currents. In contrast to the experiment that included minK, anti-fhl2 antisense had no effect on KvLQT1 alone current.

 

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  		Table 1 Voltage dependency of tail currents

 
Expression of the KvLQT1 cDNA alone generated a voltage-dependent, rapidly activating and slowly deactivating outward potassium current, as previously reported (Fig. 5D) [2–4]. Panels D–F of Fig. 5 show, that in contrast to the KvLQT1/minK/anti-fhl2 result, coexpression of the fhl2 antisense with the KvLQT1 cDNA in CHO cells had no effect on KvLQT1 currents, suggesting that the effect of fhl2 antisense cDNA is specific for IKs and dependent upon the presence of minK. The current–voltage relationship measured in Fig. 5 also remained unchanged (Fig. 5A: V1/2=9.9±7.6 mV, n=8; Fig. 5B: V1/2=18.3±3.7 mV, n=14; P=0.83. Fig. 5D: V1/2=–4.6±3.3 mV, n=10; Fig. 5E: V1/2=–8.03±1.4 mV, n=13; P=0.31). All paired current recordings were performed on the same day using cells transfected under identical conditions.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Membrane bound proteins such as ion channels are affected by a multitude of proteins during their life cycle. Well-recognized examples include cis- and/or trans-acting transcriptional and translational regulators, and function-modulating β-subunits, such as minK (reviewed in Ref. [34]). Other interacting proteins target channels to specific loci such as postsynaptic densities [35], traffic them to the membrane [36], or serve as scaffolds to anchor modulators such as kinases/phosphatases [37,38]. Indeed, preliminary data indicate that IKs too is modulated by a macromolecular signaling complex [24,39,40]. Here we identify fhl2 as a functionally important partner of the minK potassium channel subunit. This protein is enriched in heart, and interacts with minK both functionally and biochemically. Moreover, the interaction was specific for minK and did not extend to KvLQT1; we did not observe an effect of fhl2 antisense treatment on current generated by the KvLQT1 cDNA alone (Fig. 5E,F) supporting the idea of a direct interaction effect of fhl2 with minK. When anti-fhl2 antisense was coexpressed with KvLQT1 and minK, activation was still IKs-like (i.e. slow, Fig. 5, top), indicating that KvLQT1 and minK remain associated. The major effect of anti-fhl2 antisense in this experiment was a reduction in current amplitude; this result suggests that the inclusion of fhl2 in the channel complex stabilizes it at the cell surface.

Our data show that expression of IKs in HEK 293 cells, which do not contain endogenous fhl2, is dependent upon fhl2 addition. Freeman and Kass [33] have recorded IKs after minK expression in 18% of 367 transfected HEK 293 cells. However, IKs expression in this system was not dependent on the IKs {alpha} subunit KvLQT1, raising the possibility that at least a subset of these HEK cells express ion channel subunits. One study reported effects of transfected minK on the HERG protein in HEK cells [41] in the absence of exogenous fhl2, implying that minK reaches the plasma membrane without fhl2. More recently, in our lab we have been able to express IKs in HEK cells by expressing minK and KvLQT1 alone (i.e. without the requirement for cotransfection of fhl2 cDNA). Although this seemingly contradicted our previous data, we were able to reconcile the results after performing anti-fhl2 Western blots on the more recent HEK cell extracts. Indeed, we found a fhl2-related band in these extracts, indicating that HEK cells variably express a fhl2-like protein which influences expression of IKs. An important implication of this finding is that variable expression of potential ion channel modifiers by laboratory cell commonly used for electrophysiology assays may be a complicating factor in many studies.

Fhl2 is composed of cysteine-rich motifs forming Zn2+ fingers (LIM domains) thought to function as protein interaction modules in both nuclear and cytoplasmic proteins (reviewed in Ref. [16]). Li et al. [23] have shown by immunohistochemistry that fhl2 colocalizes with {alpha}-actinin at the Z-lines of human cardiac myocytes. Thus, one possible role for fhl2 is to link minK to the cytoskeleton, given that Z-lines are known to play a crucial role in the establishment and maintenance of cardiomyocyte cytoarchitecture [42]). This is consistent with an emerging body of work indicating regulation of ion channel function by interaction with the cytoskeleton [43–48]. Possible roles for compartmentalization of ion channels by cytoskeletal anchoring include the regulation of channel activity by stabilizing the protein at the cell surface, or increasing the efficiency by which intracellular signaling molecules (e.g. kinases/phosphatases) control the channel. Other LIM domain proteins have been described that influence the myocardial cytoskeletal architecture [49] or have regulatory roles in myogenic differentiation [50,51]. An accumulating body of evidence supports the view that proteins associated with the cytoskeleton serve important roles in modulating response of cardiac physiology to exogenous stress such as hypertrophy (reviewed in Ref. [52]), or drug exposure [53,54].

The generation of lines of mice homozygous for fhl2 deficiency [55,56] with no overt phenotype indicated that the gene is not essential for cardiac development and function, although in fhl2 knockouts the hypertrophic response to β-adrenergic stimulation was altered [56].

Further supporting a possible interaction between minK and fhl2 are reports showing the highest levels of fhl2 expression during murine development in cardiac septa and in the region adjacent to the atrioventricular bundle [29], a pattern which has also been noted for minK [57,58].

In summary, we have identified fhl2 as a potentially important minK interacting protein in heart. The data we report may constitute a link between the IKs channel complex and the cytoskeleton via the modular fhl2 protein. Moreover, the identification of fhl2 as an IKs modifier makes it a candidate modulator for arrhythmia syndromes such as the congenital Long QT syndrome.

Time for primary review 27 days.


   Acknowledgements
 
We would like to thank Drs Christoph Fahlke and Andrew Tapper for helpful discussions. This work was supported by an American Heart Association Scientist Development Grant to Dr Kupershmidt and grants from the United States Public Health Service (HL49989 and HL46681 to Drs Roden and Balser). Dr Roden is the holder of the William Stokes Chair in Experimental Therapeutics, a gift from the Daiichi Corporation.


   Notes
 
1 Present address: Department of Pharmacology, George Washington University, Washington, DC 20037, USA. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Xu J., Li M. Auxiliary subunits of shaker-type potassium channels. Trends Cardiovasc Med (1998) 8(5):229–234.[CrossRef][Web of Science][Medline]
  2. Barhanin J., Lesage F., Guillemare E., et al. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature (1996) 384(6604):78–80.[CrossRef][Web of Science][Medline]
  3. Sanguinetti M.C., Curran M.E., Zou A., et al. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature (1996) 384(6604):80–83.[CrossRef][Medline]
  4. Yang W.P., Levesque P.C., Little W.A., et al. KvLQT1, a voltage-gated potassium channel responsible for human cardiac arrhythmias. Proc Natl Acad Sci USA (1997) 94(8):4017–4021.[Abstract/Free Full Text]
  5. Sesti F., Goldstein S.A. Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome. J Gen Physiol (1998) 112(6):651–663.[Abstract/Free Full Text]
  6. Yang Y., Sigworth F.J. Single-channel properties of IKs potassium channels. J Gen Physiol (1998) 112(6):665–678.[Abstract/Free Full Text]
  7. Busch A.E., Busch G.L., Ford E., et al. The role of the IsK protein in the specific pharmacological properties of the IKs channel complex. Br J Pharm (1997) 122(2):187–189.[CrossRef][Web of Science][Medline]
  8. Sesti F., Tai K.K., Goldstein S.A. MinK endows the I(Ks) potassium channel pore with sensitivity to internal tetraethylammonium. Biophys J (2000) 79(3):1369–1378.[Web of Science][Medline]
  9. Splawski I., Shen J., Timothy K.W., et al. Spectrum of mutations in long-QT syndrome genes. KVLQT1, H.E.R.G. SCN5A, KCNE1, and KCNE2. Circulation (2000) 102(10):1178–1185.[Abstract/Free Full Text]
  10. Gong J., Xu J., Bezanilla M., et al. Differential stimulation of PKC phosphorylation of potassium channels by ZIP1 and ZIP2. Science (1999) 285:1565–1569.[Abstract/Free Full Text]
  11. An W.F., Bowlby M.R., Betty M., et al. Modulation of A-type potassium channels by a family of calcium sensors. Nature (2000) 403(6769):553–556.[CrossRef][Medline]
  12. Nehring R.B., Wischmeyer E., Doring F., et al. Neuronal inwardly rectifying K(+) channels differentially couple to PDZ proteins of the PSD-95/SAP90 family. J Neurosci (2000) 20(1):156–162.[Abstract/Free Full Text]
  13. Horio Y., Hibino H., Inanobe A., et al. Clustering and enhanced activity of an inwardly rectifying potassium channel, Kir4.1, by an anchoring protein, PSD-95/SAP90. J Biol Chem (1997) 272(20):12885–12888.[Abstract/Free Full Text]
  14. Sheng M., Kim E. Ion channel associated proteins. Curr Opin Neurobiol (1996) 6(5):602–608.[CrossRef][Web of Science][Medline]
  15. Chan K.K., Tsui S.K., Lee S.M., et al. Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart. Gene (1998) 210(2):345–350.[CrossRef][Web of Science][Medline]
  16. Dawid I.B., Breen J.J., Toyama R. LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet (1998) 14(4):156–162.[CrossRef][Web of Science][Medline]
  17. Way J.C., Chalfie M. mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell (1988) 54(1):5–16.[CrossRef][Web of Science][Medline]
  18. Xia H., Winokur S.T., Kuo W.L., Altherr M.R., Bredt D.S. Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs. J Cell Biol (1997) 139(2):507–515.[Abstract/Free Full Text]
  19. Nunoue K., Ohashi K., Okano I., Mizuno K. LIMK-1 and LIMK-2, two members of a LIM motif-containing protein kinase family. Oncogene (1995) 11(4):701–710.[Web of Science][Medline]
  20. Kim A.C., Peters L.L., Knoll J.H., et al. Limatin (LIMAB1), an actin-binding LIM protein, maps to mouse chromosome 19 and human chromosome 10q25, a region frequently deleted in human cancers. Genomics (1997) 46(2):291–293.[CrossRef][Web of Science][Medline]
  21. Pomies P., Louis H.A., Beckerle M.C. CRP1, a LIM domain protein implicated in muscle differentiation, interacts with alpha-actinin. J Cell Biol (1997) 139(1):157–168.[Abstract/Free Full Text]
  22. Beckerle M.C. Zyxin: zinc fingers at sites of cell adhesion. Bioessays (1997) 19(11):949–957.[CrossRef][Web of Science][Medline]
  23. Li H.Y., Kotaka M., Kostin S., et al. Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization. Cell Motil Cytoskelet (2001) 48(1):11–23.[CrossRef][Web of Science][Medline]
  24. Furukawa T., Ono Y., Tsuchiya H., et al. Specific interaction of the potassium channel beta-subunit minK with the sarcomeric protein T-cap suggests a T-tubule-myofibril linking system. J Mol Biol (2001) 313(4):775–784.[CrossRef][Web of Science][Medline]
  25. James P., Halladay J., Craig E.A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics (1996) 144(4):1425–1436.[Abstract]
  26. Schiestl R.H., Gietz R.D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet (1989) 16(5–6):339–346.[CrossRef][Web of Science][Medline]
  27. Warmke J.W., Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Nat Acad Sci USA (1994) 91(8):3438–3442.[Abstract/Free Full Text]
  28. Yang T., Wathen M.S., Felipe A., et al. K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells). Circ Res (1994) 75(5):870–878.[Abstract/Free Full Text]
  29. Chu P., Ruiz-Lozano P., Zhou Q., Cai C., Chen J. Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system. Mech Dev (2000) 95(1–2):259–265.[CrossRef][Web of Science][Medline]
  30. Demolombe S., Franco D., de Boer P., et al. Differential expression of KvLQT1 and its regulator IsK in mouse epithelia. Am J Physiol (2001) 280(2):C359–C372.[Web of Science]
  31. Sugimoto T., Tanabe Y., Shigemoto R., et al. Immunohistochemical study of a rat membrane protein which induces a selective potassium permeation: its localization in the apical membrane portion of epithelial cells. J Membr Biol (1990) 113(1):39–47.[CrossRef][Web of Science][Medline]
  32. Field L.J. Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice. Science (1988) 239:1029–1033.[Abstract/Free Full Text]
  33. Freeman L.C., Kass R.S. Expression of a minimal K+ channel protein in mammalian cells and immunolocalization in guinea pig heart. Circ Res (1993) 73(5):968–973.[Abstract/Free Full Text]
  34. Barhanin J., Attali B., Lazdunski M. IKs, a slow and intriguing cardiac K+ channel and its associated Long QT diseases. Trends Cardiovasc Med (1998) 8(5):207–214.[CrossRef][Web of Science][Medline]
  35. Kim E., Niethammer M., Rothschild A., Jan Y.N., Sheng M. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature (1995) 378(6552):85–88.[CrossRef][Medline]
  36. Bichet D., Cornet V., Geib S., et al. The I–II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit. Neuron (2000) 25(1):177–190.[CrossRef][Web of Science][Medline]
  37. Ratcliffe C.F., Qu Y., McCormick K.A., et al. A sodium channel signaling complex: modulation by associated receptor protein tyrosine phosphatase beta. Nature Neurosci (2000) 3(5):437–444.[CrossRef][Web of Science][Medline]
  38. Tsunoda S., Sierralta J., Sun Y., et al. A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature (1997) 388(6639):243–249.[CrossRef][Web of Science][Medline]
  39. Potet F., Scott J.D., Mohammad-Panah R., Escande D., Baro I. AKAP proteins anchor cAMP-dependent protein kinase to KvLQT1/IsK channel complex. Am J Physiol Heart Circ Physiol (2001) 280(5):H2038–H2045.[Abstract/Free Full Text]
  40. Marx S.O., Kurokawa J., Reiken S., et al. Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science (2002) 295(5554):496–499.[Abstract/Free Full Text]
  41. Bianchi L., Shen Z., Dennis A.T., et al. Cellular dysfunction of LQT5-minK mutants: abnormalities of IKs, IKr and trafficking in long QT syndrome. Hum Mol Genet (1999) 8(8):1499–1507.[Abstract/Free Full Text]
  42. Goncharova E.J., Kam Z., Geiger B. The involvement of adherens junction components in myofibrillogenesis in cultured cardiac myocytes. Development (1992) 114(1):173–183.[Abstract]
  43. Shin D.M., Zhao X.S., Zeng W., Mozhayeva M., Muallem S. The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity. J Cell Biol (2000) 150(5):1101–1112.[Abstract/Free Full Text]
  44. Nakahira K., Matos M.F., Trimmer J.S. Differential interaction of voltage-gated K+ channel beta-subunits with cytoskeleton is mediated by unique amino terminal domains. J Mol Neurosci (1998) 11(3):199–208.[CrossRef][Web of Science][Medline]
  45. Short D.B., Trotter K.W., Reczek D., et al. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem (1998) 273(31):19797–19801.[Abstract/Free Full Text]
  46. Gee S.H., Madhavan R., Levinson S.R., et al. Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophin-associated proteins. J Neurosci (1998) 18(1):128–137.[Abstract/Free Full Text]
  47. Maruoka N.D., Steele D.F., Au B.P., et al. Alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells. FEBS Lett (2000) 473(2):188–194.[CrossRef][Web of Science][Medline]
  48. Chauhan V.S., Tuvia S., Buhusi M., Bennett V., Grant A.O. Abnormal cardiac Na(+) channel properties and QT heart rate adaptation in neonatal ankyrin(B) knockout mice. Circ Res (2000) 86(4):441–447.[Abstract/Free Full Text]
  49. Arber S., Hunter J.J., Ross J.J., et al. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell (1997) 88(3):393–403.[CrossRef][Web of Science][Medline]
  50. Arber S., Halder G., Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell (1994) 79(2):221–231.[CrossRef][Web of Science][Medline]
  51. Fung Y.W., Wang R., Liew C.C. Characterization of a human cardiac gene which encodes for a LIM domain protein and is developmentally expressed in myocardial development. J Mol Cell Cardiol (1996) 28(6):1203–1210.[CrossRef][Web of Science][Medline]
  52. Hein S., Kostin S., Heling A., Maeno Y., Schaper J. The role of the cytoskeleton in heart failure. Cardiovasc Res (2000) 45(2):273–278.[Abstract/Free Full Text]
  53. Kolcz J., Drukala J., Jurkiewicz A., et al. Effects of cyclosporin A on contractile activity and cytoskeleton in chick embryo cardiomyocytes. Biochem Cell Biol (1999) 77(2):133–140.[CrossRef][Web of Science][Medline]
  54. Brady P.A., Alekseev A.E., Aleksandrova L.A., Gomez L.A., Terzic A. A disrupter of actin microfilaments impairs sulfonylurea-inhibitory gating of cardiac K, ATP channels. Am J Physiol (1996) 271(6):H2710–H2716.[Web of Science][Medline]
  55. Chu P.H., Bardwell W.M., Gu Y., Ross J. Jr., Chen J. FHL2 (SLIM3) is not essential for cardiac development and function. Mol Cell Biol (2000) 20(20):7460–7462.[Abstract/Free Full Text]
  56. Kong Y., Shelton J.M., Rothermel B., et al. Cardiac-specific LIM protein FHL2 modifies the hypertrophic response to beta-adrenergic stimulation. Circulation (2001) 103(22):2731–2738.[Abstract/Free Full Text]
  57. Kupershmidt S., Yang T., Anderson M.E., et al. Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system. Circ Res (1999) 84(2):146–152.[Abstract/Free Full Text]
  58. Franco D., Demolombe S., Kupershmidt S., et al. Divergent expression of delayed rectifier K(+) channel subunits during mouse heart development. Cardiovasc Res (2001) 52(1):65–75.[Abstract/Free Full Text]

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