BIOCHEMICAL CHARACTERIZATION OF hERG CHANNEL COMPOSITION IN
THE HEART
by
Rebecca Uelmen
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
(Cellular and Molecular Biology)
at the
UNIVERSITY OF WISCONSIN-MADISON
2012
Date of final oral examination: March 13, 2012
The dissertation is approved by the following members of the Final Oral Committee: Dr. Kurt Amann, Associate Professor, Zoology Dr. William Bement, Professor, Zoology Dr. Nansi Colley, Professor, Opthamology and Visual Sciences Dr. Jonathan Makielski, Professor, Department of Medicine Dr. Gail Robertson, Professor, Neuroscience i Acknowledgements
I would first like to thank my advisor, Dr. Gail Robertson, for taking me into her lab and patiently teaching me all things hERG. Under her tutelage I have grown as a scientist and a person, and for that I am deeply grateful.
Thank you to my thesis committee, Dr. Kurt Amann, Dr. Bill Bement, Dr.
Nansi Colley, Dr. Jon Makielski, and the late Dr. Paul Bertics for critical evaluation of my projects and support. It has been a privilege to learn from such great scientific minds.
Past and present members of the Robertson lab have provided lively scientific discussion and friendship throughout the years. A special thank you to
Pallavi Phartiyal, Genie Jones, Sarah Wynia-Smith, Elon Roti Roti, Sunita Joshi,
Abdalla Saad, and Fang Liu for their friendship.
The Neuroscience support staff has been very helpful with the paperwork and computer side of things. Thank you Ravi Kochhar for answering all computer questions no matter how big or small.
I am fortunate to be a student in the Cellular and Molecular Biology program, which is made exceptional by those who run the program. Thank you
Michelle Holland for going above and beyond the call of duty and providing more than academic support.
Thank you to Dr. Teresa Compton for seeing my potential as a scientist and encouraging me to pursue my Ph.D. My time with the Compton “labbies" ii showed me how fun and exciting science can be, and solidified my love of science.
I would not have made it through this process had it not been for the love and support of my family and friends. I am so fortunate to be surrounded by so many good and caring people.
Thank you to my beautiful daughter who reminds me daily of what is truly important in life, and my husband for all of his love, support, patience. I am lucky to have him in my life.
Finally, I dedicate this to my mom, whose unconditional love and support has made me the person I am today. She has always been, and will forever be, my inspiration. I am glad I was naïve enough to believe her when she said, “You can do anything you set your mind to.” I can even graduate with a Ph.D. from the
University of Wisconsin-Madison. Thanks mom.
iii Abbreviations
aLQT2 – acquired Long QT Syndrome
APD – action potential duration
Ca2+ - Calcium
C-terminus – carboxy terminus
CHO – chinese hamster ovary cells
CMC-Critical micelle concentration
CNBD – cyclic nucleotide binding domain
DN – dominant negative
ER – endoplasmic reticulum
FAK – focal adhesion kinase
FT-1 – vascular endothelial growth factor-1
HEK-293 – human embryonic kidney 293 cells
HCN – cyclic-gated nucleotide channel hERG – human ether-a-go-go related gene
Hsc – heat shock cognate
Hsp – heat shock protein
IKr – rapidly activating delayed rectifier current
IKs – slowly activating delayed rectifier current
K+ - Potassium
LQTS-Long QT Syndrome mRNA – messenger RNA iv N-terminus – amino terminus
Na+ - Sodium
NMD-nonsense-mediated decay
PH-pleckstrin homology
QTc – corrected QT interval
Sig1R – sigma 1 receptor
SR – sarcoplasmic reticulum
Tara – Trio associated repeat on actin
WT – wild type
v Table of contents
Chapter 1: Background and Significance……………….. ……………………1
1-1. Cardiac Excitability……………………………….…………………………..9 Repolarization is mediated by IKr and IKs……………………………………10 Cardiac Channelopathies……………………………………………………..10 1-2. hERG channels give rise to IKr………..…………..……………………..11 hERG structure………………………………………………………………...12 hERG Gating…………………………………………………………………...12 Native hERG channels are comprised of hERG 1a and hERG 1b…….…13 MiRP1, a putative hERG β-subunit…………………………………………..14 1-3. Congenital LQTS…………………………………………………………….15 Mechanisms of disease……………………………………………………….15 Degradation of LQT2 mutants by a NMD mechanism…………………….16 Trafficking-defective mutants give rise to LQT2……………………………16 hERG stability………………………………………………………………….17 Gating-defective mutants give rise to LQT2………………………………...18 LQT2 risk factors………………………………………………………………19 1-4. Acquired LQT2……………….……………………………………………….19 Drug block of hERG channels……………………………………………..…20 Inhibition of hERG trafficking by drugs………………………………………21 aLQT2 sex-dependence………………………………………………………21 1-5. hERG-interacting Proteins…………………..……………………………22 Proteins regulating hERG…………………………………………………….22 hERG and cancer…...…………………………………………………………24 Cytoskeletal interacting proteins………..……………………………………26 In conclusion……………………………………………………………………27 1.6. Figures and Tables………………………………………………………….29 1.7. References…………………………………………………….……………….36
Chapter 2: Expression and association of Tara with hERG…………….……46
2.1. Introduction……………………………………………………………………47 2.2. Results………………………………….………………………………………48 The carboxy-terminus of hERG directly interacts with Tara…………...…48 Expression of Native Tara in Heart…….……………….………………...... 49 Tara associates with ERG in a heterologous system and in rat and Heart……………………………………………………………………………50 Tara Localizes to M- and Z-lines and colocalizes with rERG in Rat Ventricular Myocytes………………………………………………………….51 Tara decreases hERG protein levels………………….…………………………………………………...…..52 vi
Tara decreases hERG currendensity……………………………………….52 2.3. Discussion………………..…………..………………………………………..53 2.4. Materials and Methods……………………………………………………..56 2.5. Authorship Note………………………………………………………………62 2.6. Figures…………………..………………………………………………………63 2.7. References………………….………………………………………………….75
Chapter 3: Tara may facilitate association of hERG and KCNQ1………….78
3.1. Introduction…………………….…………………………………………..…79 3.2. Results……………………..………………………………………………..…80 Confirmation of hERG and KCNQ1 association in a heterologous system and in canine ventricles…………………….…………………………………80 KCNQ1 expression and association with hERG in human ventricles.…..81 The C-termini of hERG and KCNQ1 directly interact with the C-terminus of Tara…………………………………………………….…………………….82 Tara associates with hERG but not KCNQ1 in a heterologous system ...83 Overexpression of Tara increases association of hERG and KCNQ1 ….83 Knockdown of endogenous Tara does not disrupt hERG-KCNQ1 association ………………………………………………………………….....84 3.3. Discussion……………………….…………………………………………….84 3.4. Materials and Methods….……..…………………………………………..87 3.5. Authorship Note………….………..…………………………………………91 3.6. Figures….………………….………………………………………………..…92 3.7. References………………………………………………….…………….…100
Chapter 4: hERG Isoform Expression in Human Heart…………………….……….103
4.1. Introduction……………..…………..……………………………………….104 4.2. Results……………….……………………………………………………….105 hERG expression in human heart……………...……………………………...105 hERG isoform expression in men and women…………………..…………...106 Expression of hERG 1a and 1b gycoforms in men and women……..……..107 Homogenization of human ventricular tissue……………………………..…..107 Solubilization of hERG protein from human ventricular tissue……..…..…..108 Concentration of isolated hERG protein……………...………………….…...110 Storage and stability of hERG protein………..…………..…………………...110 4.3. Discussion……………………………………………………………………110 4.4. Materials and Methods………………………..………………………….114 4.5. Authorship Note…………………………………………………………….116 4.6. Figures……………………...…………………………………………..……..117 4.7. References………………….………………………………………………..125
vii
Chapter 5: Conclusions and Future Directions………………..……………...127
5.1. Summary……………….…………………………………………………….128 5.2 Future Directions……..……….……………………….…………………..129 5.3 Closing Remarks………………….………………………………………...131
Chapter 1: Background and Significance
9 1-1. Cardiac Excitability
Mechanical pumping of the heart is achieved by the coordinated contraction of millions of myocytes by electrical impulses. Specialized pacemaker cells initiate electrical activity, which is propagated through the atria and then ventricles of the heart. This electrical activity triggers the sequential activation and inactivation of ion channels in individual myocytes to generate action potentials that begin at the surface sarcolemma and penetrate into the belly of the cell through membrane invaginations called transverse tubules (T- tubules). The action potential is divided into five phases, 0-4 (Fig. 1). Electrical stimulation from neighboring cells initiates the ventricular action potential by triggering the depolarizing influx of Na+ ions, which increases the membrane potential and triggers the opening of L-type Ca2+ channels and voltage-gated K+
+ channels. Inactivation of the Na channels and opening of transient outward (Ito) channels results in a transient repolarization seen as a “notch” in Phase 1 of the action potential. The plateau phase (Phase 2) is defined by the depolarizing influx of Ca2+ through L-type Ca2+ channels and repolarizing efflux of K+ currents.
Propagation of the action potential into T-tubules increases the intracellular Ca2+ concentration and triggers the release of additional Ca2+ from the sarcoplasmic reticulum, which binds Troponin C and exposes actin-binding sites to which myosin heads bind. This interaction produces a conformational change that initiates myofibril contraction that continues until intracellular Ca2+ is removed by re-uptake into the sarcoplasmic reticulum and expulsion through the plasma 10 membrane. The late repolarization phase, Phase 3, is mediated by the efflux of K+ ions, which repolarizes the cell and terminates the action potential (Phase
4).
Repolarization is mediated by IKr and IKs
Repolarization of the cardiac action potential was originally thought to be
+ mediated by a single K current, IK (Noble et al., 1969). Action potential recordings of guinea pig myocytes treated with the antiarrhythmic drugs d-sotalol and E-4031 showed these drugs blocked a component of IK without significantly affecting Na+ or Ca2+ currents (Sanguinetti et al., 1990). Subtraction of the blocked and unblocked currents revealed IK is composed of two distinct currents,
+ a rapidly activating K current (IKr) blocked by d-sotalol and E-4031, and an
+ unblocked slowly activating K current (IKs) (Sanguinetti et al., 1990).
Subsequent studies revealed IKs is conducted by channels encoded by KCNQ1
(KvLQT1) associated with its β-subunit, minK, and IKr by channels encoded by the human ether-a-go-go related gene (hERG/KCNH2) (Sanguinetti et al., 1995;
Trudeau et al., 1995).
Cardiac Channelopathies
The meticulous synchronization of ion channels is required to maintain proper electrical activity and heart function. Perturbation of ion channel subunits, or proteins that regulate them, give rise to “channelopathies.” One such channelopathy, Long QT Syndrome (LQTS), arises from delayed repolarization 11 of the cardiac action potential, which, as the name suggests, is reflected in prolongation of the QT interval on an electrocardiogram trace (Fig. 2). Afflicted individuals experience episodes of cardiac excitability, syncope, and sudden cardiac death due to torsade de pointe ventricular arrhythmias (Roden, 1993).
Understanding the molecular mechanisms of LQTS began in the mid-1990s with the identification of three genetic targets for ~75% of all LQTS mutations, hERG and KCNQ1, which encode K+ channels, and the SCN5A, which encodes a Na+ channel (Tester et al., 2009) (Table 1). Linkage analysis and physical mapping showed mutations in KCNQ1, hERG, and SCNA5 give rise to LQTS types 1, 2, and 3, respectively (Curran et al., 1995; Wang et al., 1995; Wang et al., 1996).
To date, 12 types of LQTS have been identified with mutations encoded in the channels themselves and ion channel regulatory proteins (Table 1).
1-2. hERG channels give rise to IKr
The human ether-a-go-go related gene (hERG) was originally identified in a human hippocampal library screen using an approach based on homology to the original ether-a-go-go isolate (Warmke and Ganetzky, 1994). High expression of the hERG transcript in heart and mapping of LQTS mutations to hERG on chromosome 7q35-36 suggested a critical role for hERG in repolarization of the cardiac action potential (Curran et al., 1995). Electrical recordings of Xenopus oocytes expressing hERG revealed biophysical and pharmacological properties nearly identical to the endogenous IKr repolarizing current found in the heart (Sanguinetti et al., 1995; Trudeau et al., 1995). These 12 functional data provided an explanation for the underlying mechanism of type
2 LQTS – loss or perturbation of cardiac IKr.
hERG structure
Analysis of the hERG sequence and hydropathy plots show hERG is structurally similar to members of the super-family of voltage-gated potassium channels (Kv), which are comprised of four α-subunits with each subunit containing six transmembrane domains (S1-S6) (MacKinnon et al., 1991;
Warmke et al., 1991) (Fig. 3A). Transmembrane domains S1-S4 form the K+ voltage sensing domain, and the S5, pore loop, and S6 domains of each subunit assemble to form the highly selective pore through which K+ ions are efficiently conducted (Fig. 3B). By inference from 3-D quaternary structures elucidated by
X-ray crystallography (Doyle et al., 1998; Long et al., 2005), hERG channels are assumed to be tetramers.
hERG Gating
In addition to structural similarities, hERG and Kv channels activate (open) and inactivate (to a non-conducting state) at depolarizing voltages, and deactivate (close) at repolarizing voltages. However, hERG differs from Kv channels in its unique rapid inactivation and slow deactivation gating properties. hERG channels are closed at negative voltages and slowly open at more positive voltages. After opening, channels quickly inactivate and become non-conducting by a C-type inactivation mechanism (Spector et al., 1996). As the cell begins to 13 repolarize and voltages become more negative, channels rapidly recover from inactivation to a conducting state and deactivate slowly, which produces a large resurgent tail current that repolarizes the cell (Sanguinetti et al., 1995; Trudeau et al., 1995) (Fig. 4).
Native hERG channels comprise 1a and 1b subunits
Most of our understanding of hERG has come from analysis of the original isolate, hERG 1a, in heterologous systems. However, increasing evidence suggests native IKr is conducted by channels comprising two α-subunits, hERG
1a, and a protein encoded by an alternately spliced transcript of the same gene, hERG 1b (Lees-Miller et al., 1997; London et al., 1997). The hERG 1a and 1b transcripts are identical except for the amino-termini, with hERG 1b being much shorter with a unique sequence (Lees-Miller et al., 1997; London et al., 1997)
(Fig. 5). Both subunits have been detected at the protein level and associate in the hearts of humans, canines, and rats, and localize to T-tubules in human myocytes (Jones et al., 2004; Wang et al., 2008). Selective knock-out of ERG 1b in mice abolishes IKr and predisposes mice to bradycardia, suggesting 1b plays an important role in native IKr, at least in mice (Lees-Miller et al., 2003). Whole cell patch clamp measurements of isolated undiseased human ventricular myocytes show current conducted by 1a/1b heteromers is more similar to native
IKr than current conducted by1a homomeric channels (O’Hara et al., 2011). In contrast to 1a, heterologous expression of 1b requires copious amounts of DNA to generate very small currents due to high retention of 1b in the ER (London et 14 al., 1997; Phartiyal et al., 2007). When coexpressed, hERG 1a and 1b associate cotranslationally and mask the 1b ER retention sites to greatly enhance 1b surface localization (Phartiyal et al., 2008). hERG 1a/1b heteromeric channels conduct nearly two-fold more current with an increased activation rate and rate of recovery from inactivation (Sale et al., 2008). To date, published data on hERG isoforms in the heart have been limited to the report of just two samples (Jones et al., 2004). In Chapter 4, I extend this analysis to additional, non-diseased human heart samples.
MiRP1, a putative hERG β-subunit
Association of hERG channels with ancillary β-subunit/s in vivo remains controversial. A study by Abbot et al. (1999) suggested hERG 1a coexpressed with MiRP1 (minK-related peptide 1/KCNE1) in Xenopus oocytes reconstituted currents with gating and conductance properties more similar to native IKr than hERG 1a alone. In addition, mutations in MiRP1 give rise to acquired and congenital LQTS (LQT6) (Abbott et al., 1999; Sesti et al., 2000; Splawski et al.,
2000). However, a direct comparison of the biophysical properties of hERG 1a +
MiRP1 in Chinese hamster ovary cells and guinea pig IKr showed coexpression of MiRP1 did not have properties closer to Ikr than hERG 1a alone (Weerapura et al., 2002). In addition to hERG, MiRP1 is reported to interact with, and modulate,
KCNQ1, cyclic-gated nucleotide channels (HCN) found in pacemaker cells, the Ito encoding α-subunit (Kv4.2), and additional channels found in neurons and rodent hearts (Tinel et al., 2000; Zhang et al., 2001; Qu et al., 2004; McCrosson 15 et al., 2009). Due to the promiscuous nature of MiRP1, understanding its role in cardiac repolarization will require additional studies in native myocytes.
1-3. Congenital LQTS
Mutations in hERG that disrupt currents give rise to congenital LQTS type
2 (LQT2), which account for 25%-30% of all LQTS cases (Tester et al., 2009)
(Table 1). Untreated, such LQTS can lead to potentially fatal torsade de pointe ventricular arrhythmia.
Mechanisms of disease
The magnitude of hERG current (I) in a myocyte is determined by the number of channels at the cell surface (N), the probability of channel opening
(Po), and the single channel current (i) (I=NPoi). In principle, disruption of hERG current by a decrease in hERG synthesis, defective trafficking through the endoplasmic reticulum (ER) and Golgi apparatus, defective gating, and/or a decrease in single channel conductance can result in a loss or reduction of hERG current and give rise to LQT2 (Deslisle et al., 2004) (Fig. 6). To date, nearly 300 hERG missense, frameshift, duplication, and deletion LQT2 mutations have been identified with missense mutations representing the majority of disease-causing mutants (www.fsm.it/cardmoc).
16 Degradation of LQT2 mutants by a nonsense-mediated decay mechanism
Most of our understanding of hERG comes from investigating the mechanisms by which LQT2 mutations cause disease. While little is known about events regulating early hERG synthesis, recent studies suggest degradation of LQT2 truncation mutant mRNA through recognition of premature stop codons by nonsense-mediated decay (NMD) (Zarraga et al., 2011; Gong et al., 2007). Expression of a LQT2 truncation mutant P926AfsX14 cDNA showed mild defects, but expression of minigenes resulted in significant reduction in mRNA, protein, and current (Zarraga et al., 2011). Of the nearly 300 LQT2 mutants identified, 25% result in premature termination, and NMD may play an important role in eliminating these defunct transcripts (www.fsm.it/cardmoc).
Trafficking-defective mutants give rise to LQT2
Trafficking of hERG channels through the ER and Golgi apparatus has been widely studied as a mechanism of LQT2 disease, and a report by Anderson et al. (2006) examining 34 LQT2 mutants showed 28 (80%) owed loss of hERG function to a trafficking defect. This is the most comprehensive analysis of LQT2 mutants to date, and suggests the majority of LQT2 mutants reduce hERG current via a trafficking defective mechanism.
Many LQT2 mutants originally reported to give rise to LQT2 via gating- defective mechanisms have more recently been shown to inhibit trafficking of hERG channels. The majority of reported gating mutants were identified in
Xenopus laevis oocytes, which are incubated at lower temperatures (~17oC). 17 Work by Zhou et al. (1999) showed rescue of trafficking-defective hERG mutants by incubation at lower temperatures (~27oC). Indeed, some mutants originally identified as gating-defective in oocytes have been shown to be trafficking mutants in mammalian cells incubated at 37oC (Anderson et al., 2006).
Trafficking of hERG proteins begins with cotranslational N-linked glycosylation in the endoplasmic reticulum (ER) identified by immunoblot as a
~135 kDa “immature” 1a band and a ~85 kDa 1b band (Zhou et al., 1998) (Fig. 7).
Proteins then traffic to the Golgi apparatus where they are terminally glycosylated and detected as ~155 kDa “mature” 1a band and ~95 kDa 1b band on immunoblots (Zhou et al., 1998; Phartiyal et al., 2007) (Fig. 7). Cleavage of the extracellular portion of hERG 1a and 1b in heterologous systems with proteinase
K suggest the majority of mature hERG is localized at the plasma membrane.
Inhibition of hERG 1a N-linked glycosylation showed glycosylation is not required for surface localization, but increases stability (Gong et al., 2002).
hERG stability
In addition to protein synthesis and maturation, stability of mature channels also contributes to the number of channels at the plasma membrane.
Recent studies have suggested exacerbation of LQTS by low K+ concentration induced by insulin, kidney, and digestive disorders is due to decreased stability of hERG channels at the plasma membrane. Rabbits fed a low K+ diet had a prolonged action potential duration attributed to a reduction in IKr (Guo et al.,
2009). Experiments conducted in a human embryonic kidney cell line (HEK-293) 18 showed that at low K+ concentrations channels entered a non-conducting state, and were rapidly internalized and degraded by the proteasome (Guo et al., 2009;
Massaeli et al., 2010). Serum starvation was used to achieve low K+ concentrations. By removing serum, the authors were not measuring low K+ in isolation, rather they were measuring the effects of hERG stability in response to serum starvation.
Gating-defective mutants give rise to LQT2
A number of mutations affecting gating have been characterized in mammalian cells incubated at physiological temperatures, and show gating defects are a mechanism of LQT2 disease. LQT2 mutants have been identified in the N- and C- termini and decrease hERG current by accelerating inactivation, prolonging recovery from inactivation, and increasing the time it takes for channels to deactivate (Sasano et al., 2004; Berecki et al., 2005; Shao et al.,
2011).
Recent studies by Gustina et al. (2011) suggest the highly conserved hERG 1a N-terminal PAS (Per Arnt Sim) domain and the carboxy-terminal cNBD
(cyclic nucleotide-binding domain) domain in 1a and 1b directly interact to regulate gating (Fig. 5). It is well established that multiple mutations in these two domains give rise to LQT2 via trafficking and gating mechanisms. Disruption of
N- and C-terminal interactions by LQT2 mutations may alter gating kinetics to decrease hERG currents.
19 LQT2 risk factors
In addition to inherited mutations, female gender is a risk factor for LQT2.
Prepubescent boys and girls have roughly the same corrected QT interval (QTc).
However, post-puberty, boys see a shortening of their QTc interval, while the
QTc interval in girls remains unchanged (Rautaharju et al., 1992). Until ~ 50 years of age the QTc interval increases in men until little difference is observed between men and women, suggesting sex hormones play a role in determining action potential duration (Merri et al., 1989; Rautaharju et al., 1992; Morcet et al.,
1999). Studies investigating athletes taking anabolic steroids and women with virilization syndrome (excessive androgen production) showed shorter QTc intervals (Stolt et al., 1999; Biodoggia et al., 2000). In contrast, castrated men had longer QTc intervals, linking testosterone to QT duration (Biodoggia et al.,
2000). A recent study shows a 62% increase in maximal hERG current in HEK-
293 cells treated with β–estradiol, suggesting hERG may have role in regulating sex-dependent differences in QT intervals (Ando et al., 2011).
1-4. Acquired LQT2
Acquired LQTS (aLQTS), a second and more clinically prevalent form of
LQTS, arises from the block of hERG channels, or inhibition of channel trafficking, by a large and diverse group of pharmaceutical drugs. Like LQT2, aLQTS puts affected individuals at risk for torsade de pointe arrhythmias and sudden cardiac death.
20 Drug block of hERG channels
Drugs that block any channel contributing to repolarization of the cardiac action potential could give rise to aLQTS. However, nearly all drugs that increase the QT interval block hERG alone (Perrin et al., 2008). The promiscuity of hERG channels is attributed to two aromatic residues at positions 652 and 656 of each subunit predicted to face the inside of the pore. Mutation of these residues to alanines abolishes the high affinity interaction of hERG channels with known hERG blockers (Mitcheson et al., 2000). Complicating identification of chemical structures that bind hERG channels is the ability of drugs to bind different combinations of residues in a single hERG subunit or in multiple subunits (Farid et al., 2006; Masetti et al., 2008; Myokai et al., 2008). The combinations of available residues is likely to change with different gating conformations, and studies suggest high-affinity hERG blockers preferentially bind the inactivated state (Ficker et al., 1998; Herzberg et al., 1998).
Recently, automated electrophysiology recordings showed fluoxetine
(Prozac) exhibits greater potency of inhibition of hERG heteromeric 1a/1b channels compared to 1a homomeric channels, while the high-affinity blockers E-
4031 and dofetilide are more potent blockers of hERG 1a homomeric channels
(Abi-Gerges et al., 2011). The unique 1a and 1b N-termini may provide different drug binding residues in homomeric versus heteromeric channels and explain the preferential binding of these drugs. This study illustrates the importance in subunit composition in estimating drug risk.
21 Inhibition of hERG trafficking by drugs
Increasing evidence suggests pharmaceutical drugs decrease IKr by two mechanisms, block of the pore and/or inhibition of hERG channel trafficking
(Wible et al., 2005; Rajamani et al., 2006; Dennis et al., 2007; Takemasa et al.,
2008; Obers et al., 2010; Han et al., 2011; Staudacher et al., 2011). Investigation of the antidepressant desipramine showed channel block increased endocytosis and degradation, and inhibited trafficking from the ER (Hong et al., 2010; Dennis et al., 2011; Staudacher et al., 2011). Perturbation of hERG by two different mechanisms may explain why some drugs are more likely to induce torsade de pointe arrhythmias and sudden cardiac death than others.
aLQTS sex-dependence
Like congenital LQT2, women are at higher risk for aLQTS than men.
Women have longer QT intervals than men, potentially predisposing them to torsade de pointe arrhythmias. In theory, any mechanism affecting modulation of adsorption, distribution, metabolism, or elimination of any hERG blocker could also contribute to aLQTS. Work by Ma et al. (2004) suggests a role for sex hormones in regulation of the CYP2J5 drug-metabolizing enzyme. Authors found that CYP2J5 levels were higher in male rats than females, but castration of male rats reduced CYP2J5 levels to that of the females. Sex-dependent differences in
CYP2J5 levels predict altered drug metabolism in male and female rats. CYP enzymes are expressed in cardiomyocytes and metabolize most IKr blockers, 22 which make them intriguing candidates for regulating sex-dependent differences in hERG drug block (Thum et al., 2000; Walles et al., 2002).
1-5. hERG-interacting Proteins
Potentially fatal diseases arise from perturbations of hERG channel synthesis, maturation, stability, and gating. Identification of the number of proteins that regulate hERG channels is increasing, with each representing a potential target for long QT syndrome.
Proteins regulating hERG
Several resident ER proteins critical in hERG maturation have been identified. The ER resident heat shock proteins (Hsp) 70 and 90, and FKB38 (38 kDa FK506-binding protein) interact with hERG and act as chaperones that regulate forward trafficking of hERG from the ER to the Golgi apparatus (Ficker et al., 2003; Walker et al., 2007). Interaction of hERG with Hsp70, Hsp 90, and
FKB38 increases maturation and current amplitude, and overexpression of
FKB38 rescues trafficking of a LQT2 mutant (Ficker et al., 2003; Walker et al.,
2007). Recently, the heat shock cognate (Hsc) 70 complexed with Hsp40 has been shown to counteract the effects of Hsp70 and decrease hERG maturation and current amplitude by increasing ubiquitination and degradation of hERG
(Walker et al., 2010; Li et al., 2011). The GM-130 (Golgi matrix, 130 kDa) protein also negatively regulates hERG trafficking and current amplitude (Roti Roti et al.,
2002). No LQTS causing mutations have been identified in chaperone proteins 23 to date, likely due to the importance of chaperones in regulating folding, unfolding, assembly, and deassembly of diverse proteins.
Recently, a role for the small GTPases Sar1 and Rab11B in trafficking and stability have been described. Overexpression of the dominant negative (DN) forms of the ER COP II export protein Sar1 and the Rab11B endosomal recycling protein significantly decreased hERG currents, golgi processing, and surface localization in a heterologous system, suggesting these small GTPases regulate
ER export in COPII vesicles and endosomal recycling prior to Golgi apparatus processing (Delisle et al., 2009).
Increasing evidence suggests hERG and KCNQ1 K+ channels associate to regulate hERG trafficking. This interaction is mediated by the C-termini of hERG and KCNQ1, with the effects of this association controversial (Ehrlich et al.,
2004; Ren et al., 2010). Work by Brunner et al. (2008) and Ren et al. (2010) showed expression of a LQT2 hERG pore mutant in CHO cells and transgenic rabbits decreased IKs in addition to IKr, and expression of a LQT1 KCNQ1 mutant decreased IKs and IKr by a trafficking mechanism, suggesting these two channels functionally interact in native systems. These results conflict with other reports in which WT KCNQ1 increased hERG trafficking and current amplitude and hERG had no effect on KCNQ1 (Ehrlich et al., 2004; Biliczki et al., 2009; Hayashi et al.,
2010).
All heterologous studies were performed in Chinese hamster ovary (CHO) cells with the decrease in hERG trafficking detected upon coexpression of
KCNQ1 fused to its β –subunit, minK, and the increase in hERG trafficking 24 observed when coexpressed with KCNQ1 alone. A report by Ehrlich et al.
(2004) showed increase of hERG current amplitude when coexpressed with
KCNQ1, and no change in hERG current amplitude when coexpressed with
KCNQ1 and minK. The interaction of hERG and KCNQ1 may have important implications for LQT. Although results are conflicting, all groups report an affect of KCNQ1 LQT1 mutants on hERG trafficking, and suggest individuals with LQT mutations that disrupt trafficking of both hERG and KCNQ1 may be at increased risk for torsade de pointe and sudden cardiac death. In Chapter 3, I describe a novel hERG-interacting protein that increases association of hERG and KCNQ1
hERG and cancer
Metastasis of cancerous cells requires proliferation and migration of cells from their place of origin to other areas of the body. Identification of hERG in several different cancerous tissues and cells lines originating from neuronal, epithelial, leukemic, and connective tissues, but not in their non-cancerous counterparts, led to the hypothesis that hERG plays a role in cancer. Indeed, an increase in hERG expression in both cancerous and non-cancerous cell lines enhances migration, and knockdown of hERG reduces cancerous cell proliferation (Lastraoili et al., 2004; Pillozzi et al., 2007; Glassmeier et al., 2012).
Block of hERG reduces proliferation in some cancer cell lines while not affecting others, suggesting enhancement of cell proliferation by hERG activation does not occur in all cancers (Pillozzi et al., 2002; Pillozzi et al., 2007; Ganapathe et al.,
2009; Plante et al., 2012). It is possible that aberrantly expressed hERG plays 25 different roles in different cancers. The involvement of hERG in migration appears at the signaling level with association and activation of hERG channels by β-1 integrins, which induces interaction of FAK (focal adhesion kinase), Rac1, and FT-1 (vascular endothelial growth factor) with hERG and β-1 integrins into a macromolecular complex that enhances cell migration (Cherubini et al., 2005;
Pillozi et al., 2007). Recent work investigating the phosphatase Ci-VSP show it is contains a voltage sensor domain that moves in response to membrane depolarization and triggers phosphatase activity (Iwaksaki et al., 2008; Sakata et al., 2011). Voltage sensing domains are highly conserved structures in ion channels, including hERG. The discovery that these domains function in signal transduction pathways independent of ion conduction through the pore provides an intriguing area of research into potential mechanisms of regulation of proliferation and migration of cancer cells by hERG.
Recently, hERG has been linked to the Sigma 1 receptor (Sig1R) of unknown function and unrelated structurally to any known proteins (Hanner et al.,
1996). Expression is limited to the brain, liver, and heart with overexpression detected in cancer cells (Spruce et al., 2004; Renaudo et al., 2004). Association of hERG with Sig1R in a myeloid leukemia cell line increases cell adhesion to fibronectin and enhances hERG currents by a trafficking mechanism (Crottes et al., 2011).
26 Cytoskeletal interacting proteins
Cytoskeletal-interacting proteins involved in the regulation of cell surface localization for several ion channels. The importance of cytoskeletal-interacting proteins in regulating ion channel function is illustrated by LQT4, which is caused by mutations in the ubiquitously expressed Ankyrin B (Mohler et al., 2003).
Ankyrins mediate attachment of integral membrane proteins, including ion channels, to the actin cytoskeleton (Mohler et al., 2003; Mohler et al., 2004a;
Lowe et al., 2008; Li et al., 2010). Ankyrin B facilitates trafficking and targeting of the Na+/Ca2+ exchanger (NCX) (Mohler et al., 2005). NCX functions to extrude
Ca2+ from the myocyte thereby depolarizing myocytes and ending cellular contraction. Disruption of NCX trafficking or membrane targeting by mutations in
Ankyrin B decreases the ability of Ca2+ to be extruded from the cell resulting in an overall net gain of Ca2+, which leads to spontaneous release of Ca2+ from the sarcoplasmic reticulum and pro-arrhythmic spontaneous myocyte contraction
(Mohler et al., 2005).
Ankyrin G coimmunoprecipitates and colocalizes with Nav1.5 sodium channels responsible for Phase 0 of the cardiac action potential in mouse and rat heart, and when knocked-down decreases Nav1.5 expression, surface localization, and Na+ current, while knock-down of Ankyrin B does not affect
Nav1.5 (Mohler et al., 2004b; Lowe et al., 2008). A Brugada Syndrome mutation in Nav1.5 disrupts association with Ankyrin G and suggests this interaction is required for normal functioning of Nav1.5 channels (Mohler et al., 2004b). 27 Like Ankyrins, filamin A is a scaffold protein that anchors transmembrane proteins to the cytoskeleton. Filamin A directly interacts with the
HCN1 (hyperpolarization-activated cation channels) pacemaker channels found in the heart and decreases currents (Gravante et al., 2004).
+ α-actinin-2 has been shown to interact and regulate Nav1.5 and Kv1.5 K channels found in pacemaker regions of the heart (Maruoka et al., 2000; Ziane et al., 2010). α-actinins perform a number of important physiological functions, including maintaining cytoskeletal organization and linking various transmembrane proteins to the actin filament network (Shulz et al., 2004; Li et al.,
2005). The Nav1.5 directly interacts with α-actinin-2, which increases surface channel density and current density (Ziane et al., 2010). In contrast, α-actinin-2 appears to decrease Kv1.5 currents since knockdown of α-actinin-2 significantly increases Kv1.5 current density (Maruoka et al., 2000).
The identification of scaffold proteins with ion channels presents a new and exciting area of research into additional mechanisms of hERG regulation and potential disease targets. In Chapter 2, I describe a novel interaction between hERG and Trio associated repeat on actin (Tara) that may underlie degradation of hERG channels.
In conclusion
The clinical importance of hERG makes full characterization of hERG channel composition and regulatory proteins an area of great interest. In particular, elucidating the role of KCNQ1 in hERG channel trafficking will facilitate 28 our understanding of LQT mechanisms. Mutations that affect association of hERG and KCNQ1 may give rise to especially malignant clinical LQT phenotypes.
Identification of proteins that mediate association of hERG and KCNQ1 may also provide additional information into mechanistic causes of LQT1 and LQT2.
Enhancement of cancer cell proliferation and migration by hERG opens up a new and exciting area of research. Block of hERG channels inhibits proliferation and migration of some cancer cells, but in others hERG block has no effect, suggesting hERG regulation by a non-conducting mechanism. Studies investigating the involvement of the hERG voltage sensing domain in signal transduction pathways may define novel functions of hERG and facilitate our understanding of cancer pathology and potential new therapies.
Investigation into block of hERG channels by pharmaceutical compounds continues to be an area of intense research. Fifty to 75 percent of lead compounds for all therapeutic targets block hERG, costing companies money and restricting the pipeline for new drugs. Elucidating mechanisms of hERG drug block and identifying characteristics of pharmaceutical compounds that induce hERG binding may open the pipeline and lead to the development of much needed drugs.
Work in this thesis adds to our understanding of hERG by examining hERG subunit composition in healthy human hearts and characterizing a novel hERG- interacting protein.
29 1.6. Figures and Tables:
Figure 1.1: Cardiac action potential
Human ventricular action potential graphed as voltage vs. time (top) and ion currents that mediate each phase of the action potential (bottom). Adapted from
Nerbonne and Kass, 2005.
Voltage
30 Figure 1.2: Prolonged QT interval on an electrocardiogram trace is a direct reflection of delayed repolarization of the action potential.
Comparison of a normal ECG trace and an abnormal ECG trace with a prolonged
QT interval. Prolongation of the QT interval is a direct measurement of the myocyte action potential duration. Adapted from Sanguinetti et al., 2006.
Normal ECG Abnormal ECG Normal action Prolonged action Trace with potential potential prolonged QT interval
Time Time
31
Table 1.1: Mutations in twelve different genes give rise to LQTS types 1-12.
The 12 different LQTS subtypes are listed in the “LQT Subtype” column, mutations in gene that give rise to LQTS in the “Mutated Gene” column, and the protein encoded by each gene and its function described in the “Encoded Protein” column. Adapted from Tester and Ackerman, 2009.
LQT Frequency Subtype Gene Encoded Protein in Patients LQT1 KCNQ1 Alpha subunit for IKs 30%-35% LQT2 hERG Alpha subunit for IKr 25%-30% LQT3 SCNA5 Alpha subunit for INa 5%-10% LQT4 ANKB Scaffold protein rare LQT5 MinK(KCNE1) Beta subunit to KCNQ1 rare Putative accessory protein for multiple LQT6 MiRP1(KCNE2) ion channels rare LQT7 KCNJ2 Alpha subunit for IK1 rare LQT8 CACNA1C Alpha subunit for ICa rare LQT9 CAV3 Caveolin-3 - Nav1.5-interacting protein rare LQT10 SCN4B Navβ4 Beta subunit for Ina rare
LQT11 AKAP9 Yotiao - IKs scaffold protein rare Syntrophin-α1 - Nav1.5 ineracting LQT12 SNTA1 protein rare
32 Figure 1.3: Tetrameric assembly of hERG subunits
A. Diagram of a single hERG subunit containing six α-helical transmembrane domains, S1–S6. B. Tetrameric assembly of hERG α-subunits form channel with
S5, S6, and the pore loop forming the highly selective K+ pore. Adapted from
Nerbonne and Kass, 2005.
A B
Figure 1.4: Gating kinetics of hERG channels.
Channels are closed at negative voltages. Membrane depolarization slowly activates (opens) the channels, which then inactivate rapidly, especially at higher potentials. Repolarization of the membrane reverses the transitions between these channels states. C-type inactivation is thought to be caused by constriction of the selectivity filter (circled in red). Adapted from Sanguinetti, 2006.
33 Figure 1.5: Primary structure of hERG 1a and 1b.
Graphic showing N-terminal differences in structure between hERG 1a (A) and hERG 1b (B). Cytoplasmic N and C termini flank the hydrophobic core. N- terminal differences yield subunits of 1159- and 819-aa residues for hERG 1a and 1b, respectively. PAS indicates Per–Arnt–Sim domain; PAC, PAS- associated C-terminal domain; CNBD, putative cyclic nucleotide binding domain;
TCC, tetramerization coiled-coil domain. Adapted from Sale et al., 2008.
34 Figure 1.6: Mechanisms of LQT2.
The magnitude of hERG current is defined by I=NPoi, where N=the number of channels at the cell surface, Po the probability of channel opening, and i, the current conducted by individual channels. Mutations in hERG can give rise to
LQT2 by decreasing the number of channels at the cell surface or disruption of gating and/or conductance.
35 Figure 1.7: hERG immunoblot of HEK-293 cells expressing hERG 1a and 1b.
Mature hERG 1a migrates at ~155 kDa, immature 1a ~135 kDa, mature 1b ~95 kDa, immature 1b ~85, and unglycosylated 1b ~80 kDa.
36 1.7. References
Abi-Gerges N, Holkham H, Jones EM, Pollard CE, Valentin JP, Robertson GA (2011). hERG subunit composition determines differential drug sensitivity. Br J Pharmacol. 164(2b):419-32. Abbott GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating MT, Goldstein SA (1999). MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell. 97(2):175-87. Anderson CL, Delisle BP, Anson BD, Kilby JA, Will ML, Tester DJ, Gong Q, Zhou Z, Ackerman MJ, January CT (2006). Most LQT2 mutations reduce Kv11.1 (hERG) current by a class 2 (trafficking-deficient) mechanism. Circulation. 113(3):365-73. Ando F, Kuruma A, Kawano S (2011). Synergic effects of β-estradiol and erythromycin on hERG currents. J Membr Biol. 241(1):31-8. Berecki G, Zegers JG, Verkerk AO, Bhuiyan ZA, de Jonge B, Veldkamp MW, Wilders R, van Ginneken AC (2005). HERG channel (dys)function revealed by dynamic action potential clamp technique. Biophys J. 88(1):566-78. Bidoggia H, Maciel JP, Capalozza N, Mosca S, Blaksley EJ, Valverde E, Bertran G, Arini P, Biagetti MO, Quinteiro RA (2000). Sex-dependent electrocardiographic pattern of cardiac repolarization. Am Heart J. 140(3):430-6. Biliczki P, Girmatsion Z, Brandes RP, Harenkamp S, Pitard B, Charpentier F, Hébert TE, Hohnloser SH, Baró I, Nattel S, Ehrlich JR (2009). Trafficking- deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction. Heart Rhythm. 6(12):1792- 801. Brunner M, Peng X, Liu GX, Ren XQ, Ziv O, Choi BR, Mathur R, Hajjiri M, Odening KE, Steinberg E, Folco EJ, Pringa E, Centracchio J, Macharzina RR, Donahay T, Schofield L, Rana N, Kirk M, Mitchell GF, Poppas A, Zehender M, Koren G (2008). Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome. J Clin Invest. 118(6):2246-59. Cherubini A, Hofmann G, Pillozzi S, Guasti L, Crociani O, Cilia E, Di Stefano P, Degani S, Balzi M, Olivotto M, Wanke E, Becchetti A, Defilippi P, Wymore R, Arcangeli A (2005). Human ether-a-go-go-related gene 1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signaling. Mol Biol Cell. 16(6):2972-83. Crottès D, Martial S, Rapetti-Mauss R, Pisani DF, Loriol C, Pellissier B, Martin P, Chevet E, Borgese F, Soriani O (2011). Sig1R protein regulates hERG channel expression through a post-translational mechanism in leukemic cells. J Biol Chem. 286(32):27947-58.
37 Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT (1995). A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80(5): 795-803.
Delisle BP, Anson BD, Rajamani S, January CT (2004). Biology of cardiac arrhythmias: ion channel protein trafficking. Circ Res. 94(11):1418-28. Delisle BP, Underkofler HA, Moungey BM, Slind JK, Kilby JA, Best JM, Foell JD, Balijepalli RC, Kamp TJ, January CT (2009). Small GTPase determinants for the Golgi processing and plasmalemmal expression of human ether-a-go-go related (hERG) K+ channels. J Biol Chem. 284(5):2844-53. Dennis A, Wang L, Wan X, Ficker E (2007). hERG channel trafficking: novel targets in drug-induced long QT syndrome. Biochem Soc Trans. 35(Pt 5):1060-3. Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998). The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280(5360):69-77. Ehrlich JR, Pourrier M, Weerapura M, Ethier N, Marmabachi AM, Hébert TE, Nattel S (2004). KvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currents. J Biol Chem. 279(2):1233-41. Farid R, Day T, Friesner RA, Pearlstein RA (2006). New insights about HERG blockade obtained from protein modeling, potential energy mapping, and docking studies. Bioorg Med Chem. 14(9):3160-73. Ficker E, Jarolimek W, Kiehn J, Baumann A, Brown AM (1998). Molecular determinants of dofetilide block of HERG K+ channels. Circ Res. 82(3):386-95. Ficker E, Dennis AT, Wang L, Brown AM (2003). Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERG. Circ Res. 92(12):e87-100. Ganapathi SB, Kester M, Elmslie KS (2009). State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy. Am J Physiol Cell Physiol. 296(4):C701-10. Glassmeier G, Hempel K, Wulfsen I, Bauer CK, Schumacher U, Schwarz JR (2012). Inhibition of HERG1 K(+) channel protein expression decreases cell proliferation of human small cell lung cancer cells. Pflugers Arch. 463(2):365-76. Gong Q, Anderson CL, January CT, Zhou Z (2002). Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol. 283(1):H77-84. Gong Q, Zhang L, Vincent GM, Horne BD, Zhou Z (2007). Nonsense mutations in hERG cause a decrease in mutant mRNA transcripts by nonsense-mediated mRNA decay in human long-QT syndrome. Circulation. 116(1):17-24. Gravante B, Barbuti A, Milanesi R, Zappi I, Viscomi C, DiFrancesco D (2004). 38 Interaction of the pacemaker channel HCN1 with filamin A. J Biol Chem. 279(42):43847-53. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, Zhang S (2009). Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. J Clin Invest. 119(9):2745-57. Gustina AS, Trudeau MC (2011). hERG potassium channel gating is mediated by N- and C-terminal region interactions. J Gen Physiol. 137(3):315-25.
Han S, Zhang Y, Chen Q, Duan Y, Zheng T, Hu X, Zhang Z, Zhang L (2011). Fluconazole inhibits hERG K(+) channel by direct block and disruption of protein trafficking. Eur J Pharmacol. 650(1):138-44. Hayashi K, Shuai W, Sakamoto Y, Higashida H, Yamagishi M, Kupershmidt S (2010). Trafficking-competent KCNQ1 variably influences the function of HERG long QT alleles. Heart Rhythm. 7(7):973-80. Herzberg IM, Trudeau MC, Robertson GA (1998). Transfer of rapid inactivation and sensitivity to the class III antiarrhythmic drug E-4031 from HERG to M-eag channels. J Physiol. 511 ( Pt 1):3-14. Hong HK, Park MH, Lee BH, Jo SH (2010). Block of the human ether-a-go-go- related gene (hERG) K+ channel by the antidepressant desipramine. Biochem Biophys Res Commun. 394(3):536-41. Iwasaki H, Murata Y, Kim Y, Hossain MI, Worby CA, Dixon JE, McCormack T, Sasaki T, Okamura Y (2008). A voltage-sensing phosphatase, Ci-VSP, which shares sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5- bisphosphate. Proc Natl Acad Sci U S A.105(23):7970-5. Jones EM, Roti Roti EC, Wang J, Delfosse SA, Robertson GA (2004). Cardiac IKr channels minimally comprise hERG 1a and 1b subunits. J Biol Chem. 279(43):44690-4.
Kapplinger JD, Tester DJ, Alders M, Benito B, Berthet M, Brugada J, et al. (2010). An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing. Heart Rhythm 7(1): 33-46.
Kitajiri S, Sakamoto T, Belyantseva IA, Goodyear RJ, Stepanyan R, Fujiwara I, Bird JE, Riazuddin S, Riazuddin S, Ahmed ZM, Hinshaw JE, Sellers J, Bartles JR, Hammer JA 3rd, Richardson GP, Griffith AJ, Frolenkov GI, Friedman TB (2010). Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing. Cell. 141(5):786-98. Lastraioli E, Guasti L, Crociani O, Polvani S, Hofmann G, Witchel H, Bencini L, Calistri M, Messerini L, Scatizzi M, Moretti R, Wanke E, Olivotto M, Mugnai G, Arcangeli A (2004). herg1 gene and HERG1 protein are overexpressed in colorectal cancers and regulate cell invasion of tumor cells. Cancer Res. 39 64(2):606-11. Lees-Miller JP, Kondo C, Wang L, Duff HJ (1997). Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circulation Research 81(5):719-26.
Lees-Miller JP, Guo J, Somers JR, Roach DE, Sheldon RS, Rancourt DE, Duff HJ (2003). Selective knockout of mouse ERG1 B potassium channel eliminates I(Kr) in adult ventricular myocytes and elicits episodes of abrupt sinus bradycardia. Mol Cell Biol. 23(6):1856-62. Li J, Kline CF, Hund TJ, Anderson ME, Mohler PJ (2010). Ankyrin-B regulates Kir6.2 membrane expression and function in heart. J Biol Chem. 285(37):28723- 30.
Li P, Ninomiya H, Kurata Y, Kato M, Miake J, Yamamoto Y, Igawa O, Nakai A, Higaki K, Toyoda F, Wu J, Horie M, Matsuura H, Yoshida A, Shirayoshi Y, Hiraoka M, Hisatome I (2011). Reciprocal control of hERG stability by Hsp70 and Hsc70 with implication for restoration of LQT2 mutant stability. Circ Res. 108(4):458-68. Li Q, Montalbetti N, Shen PY, Dai XQ, Cheeseman CI, Karpinski E, Wu G, Cantiello HF, Chen XZ (2005). Alpha-actinin associates with polycystin-2 and regulates its channel activity. Hum Mol Genet. 14(12):1587-603. London B, Trudeau MC, Newton KP, Beyer AK, Copeland NG, Gilbert DJ, Jenkins NA, Satler CA, Robertson GA (1997). Two isoforms of the mouse ether- a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circulation Research 81(5):870-8.
Long SB, Campbell EB, Mackinnon R (2005). Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science. 309(5736):897-903. Epub 2005 Jul 7.
Lowe JS, Palygin O, Bhasin N, Hund TJ, Boyden PA, Shibata E, Anderson ME, Mohler PJ (2008). Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway. J Cell Biol. 180(1):173-86. Ma J, Graves J, Bradbury JA, Zhao Y, Swope DL, King L, Qu W, Clark J, Myers P, Walker V, Lindzey J, Korach KS, Zeldin DC (2004). Regulation of mouse renal CYP2J5 expression by sex hormones. Mol Pharmacol. 65(3):730-43. MacKinnon R (1991). Determination of the subunit stoichiometry of a voltage- activated potassium channel. Nature 350(6315):232-5.
Maruoka ND, Steele DF, Au BP, Dan P, Zhang X, Moore ED, Fedida D (2000). alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and 40 channel localization in HEK cells. FEBS Lett. 473(2):188-94. Massaeli H, Guo J, Xu J, Zhang S (2010). Extracellular K+ is a prerequisite for the function and plasma membrane stability of HERG channels. Circ Res. 106(6):1072-82. Masetti M, Cavalli A, Recanatini M (2008). Modeling the hERG potassium channel in a phospholipid bilayer: Molecular dynamics and drug docking studies. J Comput Chem. 29(5):795-808. McCrossan ZA, Roepke TK, Lewis A, Panaghie G, Abbott GW (2009). Regulation of the Kv2.1 potassium channel by MinK and MiRP1. J Membr Biol. 228(1):1-14. Merri M, Benhorin J, Alberti M, Locati E, Moss AJ (1989). Electrocardiographic quantitation of ventricular repolarization. Circulation. 80(5):1301-8. Mitcheson JS, Chen J, Lin M, Culberson C, Sanguinetti MC (2000). A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci. 97(22):12329-33. Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogné K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, Bennett V (2003). Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature. 421(6923):634-9. Mohler PJ, Splawski I, Napolitano C, Bottelli G, Sharpe L, Timothy K, Priori SG, Keating MT, Bennett V (2004a). A cardiac arrhythmia syndrome caused by loss of ankyrin-B function. Proc Natl Acad Sci. 101(24):9137-42. Mohler, P.J., I. Rivolta, C. Napolitano, G. Lemaillet, S. Lambert, S.G. Priori, and V. Bennett (2004b). Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes. Proc. Natl. Acad. Sci. USA. 101:17533–17538. Mohler PJ, Davis JQ, Bennett V (2005). Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain. PLoS Biol. 3(12):e423. Morcet JF, Safar M, Thomas F, Guize L, Benetos A (1999). Associations between heart rate and other risk factors in a large French population. J Hypertens. 17(12 Pt 1):1671-6. Myokai T, Ryu S, Shimizu H, Oiki S (2008). Topological mapping of the asymmetric drug binding to the human ether-à-go-go-related gene product (HERG) potassium channel by use of tandem dimers. Mol Pharmacol. 73(6):1643-51. Nerbonne JM, Kass RS (2005). Molecular physiology of cardiac repolarization. Physiol Rev. 85(4):1205-53. Noble D, Tsien RW (1969). Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres. J Physiol 200(1): 205-231. 41
Obers S, Staudacher I, Ficker E, Dennis A, Koschny R, Erdal H, Bloehs R, Kisselbach J, Karle CA, Schweizer PA, Katus HA, Thomas D (2010). Multiple mechanisms of hERG liability: K+ current inhibition, disruption of protein trafficking, and apoptosis induced by amoxapine. Naunyn Schmiedebergs Arch Pharmacol. 381(5):385-400. O'Hara T, Virág L, Varró A, Rudy Y (2011). Simulation of the undiseased human cardiac ventricular action potential: model formulation and experimental validation. PLoS Comput Biol. 7(5):e1002061.
Perrin MJ, Subbiah RN, Vandenberg JI, Hill AP (2009). Human ether-a-go-go related gene (hERG) K+ channels: function and dysfunction. Prog Biophys Mol Biol. 98(2-3):137-48.
Phartiyal P, Jones EM, Robertson GA (2007). Heteromeric assembly of human ether-à-go-go-related gene (hERG) 1a/1b channels occurs cotranslationally via N-terminal interactions. J Biol Chem. 282(13):9874-82.
Phartiyal P, Sale H, Jones EM, Robertson GA (2008). Endoplasmic reticulum retention and rescue by heteromeric assembly regulate human ERG 1a/1b surface channel composition. J Biol Chem. 283(7):3702-7.
Pillozzi S, Brizzi MF, Balzi M, Crociani O, Cherubini A, Guasti L, Bartolozzi B, Becchetti A, Wanke E, Bernabei PA, Olivotto M, Pegoraro L, Arcangeli A (2002). HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors. Leukemia. 16(9):1791-8.
Pillozzi S, Brizzi MF, Bernabei PA, Bartolozzi B, Caporale R, Basile V, Boddi V, Pegoraro L, Becchetti A, Arcangeli A (2007). VEGFR-1 (FLT-1), beta1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome. Blood. 110(4):1238-50. Plante I, Vigneault P, Drolet B, Turgeon J (2012). Rosuvastatin blocks hERG current and prolongs cardiac repolarization. J Pharm Sci. 101(2):868-78. Qu J, Kryukova Y, Potapova IA, Doronin SV, Larsen M, Krishnamurthy G, Cohen IS, Robinson RB (2004). MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes. J Biol Chem. 279(42):43497-502. Rajamani S, Eckhardt LL, Valdivia CR, Klemens CA, Gillman BM, Anderson CL, Holzem KM, Delisle BP, Anson BD, Makielski JC, January C (2006). Drug- induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine. Br J Pharmacol. 149(5):481-9. Rautaharju PM, Zhou SH, Wong S, Calhoun HP, Berenson GS, Prineas R, 42 Davignon A (1992). Sex differences in the evolution of the electrocardiographic QT interval with age. Can J Cardiol. 8(7):690-5. Ren XQ, Liu GX, Organ-Darling LE, Zheng R, Roder K, Jindal HK, Centracchio J, McDonald TV, Koren G (2010). Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines. Am J Physiol Heart Circ Physiol. 299(5):H1525-34. Renaudo A, Watry V, Chassot AA, Ponzio G, Ehrenfeld J, Soriani O (2004). Inhibition of tumor cell proliferation by sigma ligands is associated with K+ Channel inhibition and p27kip1 accumulation. J Pharmacol Exp Ther. 311(3):1105-14. Riazuddin S, Khan SN, Ahmed ZM, Ghosh M, Caution K, Nazli S, et al. (2006). Mutations in TRIOBP, Which Encodes a Putative Cytoskeletal-Organizing Protein, Are Associated with Nonsyndromic Recessive Deafness. Am J Hum Genet 78(1): 137-143.
Riazuddin S KS, Ahmed ZM, Ghosh M, Caution K, Nazli S, Kabra M, Zafar AU, Chen K, Naz S, Antonellis A, Pavan WJ, Green ED, Wilcox ER, Firedman PL, Morell RJ, Riazuddin S, and Friedman TB. (2006). Mutations in TRIOBP, Which Encodes a Putative Cytoskeletal-Organizing Protein, Are Associated with Nonsyndromic Recessive Deafness. American Journal of Human Genetics 78: 137-143.
Roden DM (1993). Early after-depolarizations and torsade de pointes: implications for the control of cardiac arrhythmias by prolonging repolarization. Eur Heart J 14 Suppl H: 56-61.
Roti EC, Myers CD, Ayers RA, Boatman DE, Delfosse SA, Chan EK, Ackerman MJ, January CT, Robertson GA (2002). Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene. J Biol Chem. 277(49):47779-85.
Sakata S, Hossain MI, Okamura Y (2011). Coupling of the phosphatase activity of Ci-VSP to its voltage sensor activity over the entire range of voltage sensitivity. J Physiol. 589(Pt 11):2687-705.
Sale H, Wang J, O'Hara TJ, Tester DJ, Phartiyal P, He JQ, Rudy Y, Ackerman MJ, Robertson GA (2008). Physiological properties of hERG 1a/1b heteromeric currents and a hERG 1b-specific mutation associated with Long-QT syndrome. Circ Res. 103(7):e81-95.
Sanguinetti MC, Jurkiewicz NK (1990). Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 96(1): 195-215.
43 Sanguinetti MC, Jiang C, Curran ME, Keating MT (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81(2): 299-307.
Sanguinetti MC, Tristani-Firouzi (2006). hERG potassium channels and cardiac arrhythmia. Nature. 440(7083):463-9. Sasano T, Ueda K, Orikabe M, Hirano Y, Kawano S, Yasunami M, Isobe M, Kimura A, Hiraoka M (2004). Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation. J Mol Cell Cardiol. 37(6):1205-11. Seipel K, O'Brien SP, Iannotti E, Medley QG, Streuli M (2001). Tara, a novel F- actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization. J Cell Sci 114(Pt 2): 389-399.
Sesti F, Abbott GW, Wei J, Murray KT, Saksena S, Schwartz PJ, Priori SG, Roden DM, George AL Jr, Goldstein SA (2000). A common polymorphism associated with antibiotic-induced cardiac arrhythmia. Proc Natl Acad Sci. 97(19):10613-8.
Shahin H, Walsh T, Sobe T, Abu Sa'ed J, Abu Rayan A, Lynch ED, et al. (2006). Mutations in a novel isoform of TRIOBP that encodes a filamentous-actin binding protein are responsible for DFNB28 recessive nonsyndromic hearing loss. Am J Hum Genet 78(1): 144-152.
Shao C, Lu Y, Liu M, Chen Q, Lan Y, Liu Y, Lin M, Li Y (2011). Electrophysiological study of V535M hERG mutation of LQT2. J Huazhong Univ Sci Technolog Med Sci. 31(6):741-8. Spector PS, Curran ME, Zou A, Keating MT, Sanguinetti MC (1996). Fast inactivation causes rectification of the IKr channel J Gen Physiol. 107(5):611-9.
Splawski I, Shen J, Timothy KW, Lehmann MH, Priori S, Robinson JL, Moss AJ, Schwartz PJ, Towbin JA, Vincent GM, Keating MT (2000). Spectrum of mutations in long-QT syndrome genes. KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation. 102(10):1178-85.
Spruce BA, Campbell LA, McTavish N, Cooper MA, Appleyard MV, O'Neill M, Howie J, Samson J, Watt S, Murray K, McLean D, Leslie NR, Safrany ST, Ferguson MJ, Peters JA, Prescott AR, Box G, Hayes A, Nutley B, Raynaud F, Downes CP, Lambert JJ, Thompson AM, Eccles S (2004). Small molecule antagonists of the sigma-1 receptor cause selective release of the death program in tumor and self-reliant cells and inhibit tumor growth in vitro and in vivo. Cancer Res. 64(14):4875-86. Staudacher I, Wang L, Wan X, Obers S, Wenzel W, Tristram F, Koschny R, 44 Staudacher K, Kisselbach J, Koelsch P, Schweizer PA, Katus HA, Ficker E, Thomas D (2011). hERG K+ channel-associated cardiac effects of the antidepressant drug desipramine. Naunyn Schmiedebergs Arch Pharmacol. 383(2):119-39. Stolt A, Karila T, Viitasalo M, Mäntysaari M, Kujala UM, Karjalainen J (1999). QT interval and QT dispersion in endurance athletes and in power athletes using large doses of anabolic steroids. Am J Cardiol. 84(3):364-6. Sugaya M, Takenoyama M, Shigematsu Y, Baba T, Fukuyama T, Nagata Y, Mizukami M, So T, Ichiki Y, Yasuda M, So T, Hanagiri T, Sugio K, Yasumoto K (2007). Identification of HLA-A24 restricted shared antigen recognized by autologous cytotoxic T lymphocytes from a patient with large cell carcinoma of the lung. Int J Cancer. 120(5):1055-62. Takemasa H, Nagatomo T, Abe H, Kawakami K, Igarashi T, Tsurugi T, Kabashima N, Tamura M, Okazaki M, Delisle BP, January CT, Otsuji Y (2008). Coexistence of hERG current block and disruption of protein trafficking in ketoconazole-induced long QT syndrome. Br J Pharmacol. 153(3):439-47. Tester DJ, Ackerman MJ (2009). Cardiomyopathic and channelopathic causes of sudden unexplained death in infants and children. Annu Rev Med 60: 69-84.
Thum T, Borlak J (2000). Cytochrome P450 mono-oxygenase gene expression and protein activity in cultures of adult cardiomyocytes of the rat. Br J Pharmacol. 130(8):1745-52. Tinel N, Diochot S, Lauritzen I, Barhanin J, Lazdunski M, Borsotto M (2000). M- type KCNQ2-KCNQ3 potassium channels are modulated by the KCNE2 subunit. FEBS Lett. 480(2-3):137-41.
Trudeau MC, Warmke JW, Ganetzky B, Robertson GA (1995). HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269(5220): 92-95.
Walker VE, Atanasiu R, Lam H, Shrier A (2007). Co-chaperone FKBP38 promotes HERG trafficking. J Biol Chem. 282(32):23509-16. Walker VE, Wong MJ, Atanasiu R, Hantouche C, Young JC, Shrier A (2010). Hsp40 chaperones promote degradation of the HERG potassium channel. J Biol Chem. 285(5):3319-29. Walles M, Thum T, Levsen K, Borlak J (2002). Verapamil: new insight into the molecular mechanism of drug oxidation in the human heart. J Chromatogr A. 970(1-2):117-30. Wang Q, Shen J, Splawski I, Atkinson D, Li Z, Robinson JL, et al. (1995). SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 80(5): 805-811.
45 Wang DW, Yazawa K, George AL Jr, Bennett PB (1996). Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome. Proc Natl Acad Sci. 93(23):13200-5. Wang X, Xu R, Abernathey G, Taylor J, Alzghoul MB, Hannon K, Hockerman GH, Pond AL (2008). Kv11.1 channel subunit composition includes MinK and varies developmentally in mouse cardiac muscle. Dev Dyn. 237(9):2430-7.
Warmke JW, Ganetzky B (1994). Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome. Proc Natl Acad Sci. 91(8):3438-42. Weerapura M, Nattel S, Chartier D, Caballero R, Hébert TE (2002). A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link? J Physiol. 540(Pt 1):15-2. Wible BA, Hawryluk P, Ficker E, Kuryshev YA, Kirsch G, Brown AM (2005). HERG-Lite: a novel comprehensive high-throughput screen for drug-induced hERG risk. J Pharmacol Toxicol Methods. 52(1):136-45. Yano T, Yamazaki Y, Adachi M, Okawa K, Fort P, Uji M, Tsukita S, Tsukita S (2011). Tara up-regulates E-cadherin transcription by binding to the Trio RhoGEF and inhibiting Rac signaling. J Cell Biol. 193(2):319-32. Zarraga IG, Zhang L, Stump MR, Gong Q, Vincent GM, Zhou Z (2011). Nonsense-mediated mRNA decay caused by a frameshift mutation in a large kindred of type 2 long QT syndrome. Heart Rhythm. 8(8):1200-6. Zhang M, Jiang M, Tseng GN (2001). minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel? Circ Res. 88(10):1012-9. Ziane R, Huang H, Moghadaszadeh B, Beggs AH, Levesque G, Chahine M (2010). Cell membrane expression of cardiac sodium channel Na(v)1.5 is modulated by alpha-actinin-2 interaction. Biochemistry. 49(1):166-78. Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA, January C (1998). Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J. 74(1):230-41. Zhou Z, Gong Q, January CT (1999). Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome. Pharmacological and temperature effects. J Biol Chem. 274(44):31123-6.
Ziane R, Huang H, Moghadaszadeh B, Beggs AH, Levesque G, Chahine M (2010). Cell membrane expression of cardiac sodium channel Na(v)1.5 is modulated by alpha-actinin-2 interaction. Biochemistry. 49(1):166-78.
46
Chapter 2: Expression and association of Tara with hERG
47 2.1. Introduction
Long QT Syndrome is a cardiac disorder characterized by a prolonged QT interval on the surface electrocardiogram with clinical symptoms that range from episodes of cardiac excitability, syncope, torsade de pointe, and, in severe cases, sudden cardiac death (Curran et al., 1995). Twelve different LQTS genes have been identified with one of the most prominent forms linked to mutations in voltage-gated potassium channels encoded by the human ether-a-go-go related gene (hERG/KCNH2), which give rise to the repolarizing current IKr in the heart
(Sanguinetti et al., 1995; Trudeau et al., 1995). Functional expression studies of
LQTS mutants have revealed multiple mechanisms responsible for loss of hERG current, including abnormalities in hERG synthesis and protein trafficking (Zhou et al., 1998; Anderson et al., 2006). While over 300 type 2 LQTS mutants have been identified (http://www.fsm.it/cardmoc), little is known about protein complexes that facilitate hERG synthesis, folding, maturation, and retention.
Each component of such complexes represents a potential target for LQTS.
To identify novel proteins involved in the hERG life cycle, we carried out a yeast two-hybrid screen to look for human cardiac proteins that interact with the
C-terminal region of hERG. Here we report Trio associated repeat on actin (Tara), interacts with hERG to decrease protein expression. Previously, Tara was identified as a ubiquitously expressed Trio binding protein that directly bound and stabilized F-actin when expressed heterologously (Seipel et al., 2001; Riazuddin et al., 2006; Shahin et al., 2006). By using yeast two-hybrid interaction assays, 48 immunocytochemistry, biochemistry, and electrophysiology, we found that hERG interacts with bands recognized by Tara antibodies in a heterologous system and in rat myocytes. Overexpression of Tara with hERG decreased hERG protein expression in HEK-293 cells, which correlated to a decrease in amplitude of current expressed in oocytes. We propose that the Tara plays a previously undefined role in modulation of the hERG life cycle.
2.2. Results
The carboxy-terminus of hERG directly interacts with Tara
In an effort to identify novel hERG interacting proteins, we conducted a two- hybrid screen of a human heart cDNA library using the carboxy-terminus (C- terminus) of hERG (amino acids 669-1159) as bait (Roti Roti et al. 2002) (Fig. 1).
Of 2.2x106 clones screened, five hERG binding partners were identified, one of which mapped to Trio associate repeat on actin (Tara). Two independent clones corresponding to amino acids 314-593 and 361-593 of the coiled-coil rich C- terminus of Tara directly bound hERG (Table 1). The interactions appeared specific to hERG as the Tara fragments failed to bind the C-terminus of Shaker B, a distantly related potassium channel expressed in Drosophila (Papazian et al.,
1987). Binary 2-hybrid tests confirmed the association of Tara’s C-terminal fragments with the C-terminus of hERG, and showed a direct association of full- length Tara with hERG. 49
Expression of Native Tara in Heart
Antibodies targeting different epitopes were used to characterize Tara in human, rat, and dog heart ventricles (Fig. 2). Tara immunoblots of protein preparations from human heart ventricles revealed a 70-73 kDa band in 10 of 10 blots (Fig. 2A). Although additional bands were detected (Table 2), this is the only band that was consistently identified, and which corresponds to Tara’s predicted size of 72 kDa in humans. Moreover, a similarly-sized band of 70-72 kDa identified in human-derived HEK-293 cells was selectively knocked down by
Tara siRNA (Fig. 2B and C). Of the additional bands detected, only the 119 kDa band was detected in 3 of 3 blots, and knockdown of Tara did not significantly alter its expression levels. Together, these results suggest Tara migrates as an
~72 kDa band in human tissue. In 7 of 7 rat hearts and 8 of 9 dog hearts, Tara immunoblots revealed a band of 65-68 kDa (Fig. 2D and E). While additional bands were detected (Tables 3 and 4), only the 65-68 kDa band was detected by two different Tara antibodies, and which corresponds to the predicted size of
Tara in rat. Tara expression has not been examined in dog, and these results suggest Tara is expressed in dog and rat hearts and migrates as a 65-68 kDa band.
50 Bands corresponding to Tara associate with ERG in a heterologous system and in rat and heart.
We next used coimmunoprecipitation to determine whether hERG and
Tara associate in situ in HEK-293 cells. Consistent with predictions from western blot analysis of Tara described above, the anti-hERG antibody immunoprecipitated a 72 kDa detected by a Tara antibody (Fig. 3A). In approximately 50% of experiments, a band of 75 kDa was also detected, which had not been seen in Tara immunoblots of input lysates. Tara’s association appeared specific for hERG since overexpressed Tara did not associate with
KCNQ1 (See Chapter 3). I conducted complementary experiments in which
HEK-293 cells stably expressing hERG 1a were immunoprecipitated with an anti-
Tara antibody and immunoblotted for hERG. These blots showed proteins immunoprecipitated by the Tara antibody associate with both mature and immature hERG 1a (Fig. 3B). The bi-directional coimmunoprecipitation supports the conclusion that Tara and hERG exhibit a bona fide interaction in cells. This interaction occurred with low efficiency, judging by the robust signal remaining in the flow-through (see Discussion).
A corresponding association was observed in native tissue. We used hERG antibodies to immunoprecipitate rat ERG (rERG) and immunoblotted with
Tara antibodies. Integral membrane protein preparations from rat ventricles revealed association of rERG with a 65-68 kDa band detected by the Tara antibody, corresponding to the signal observed in rat heart lysates (Fig. 3C).
This association was confirmed using two different antibodies to 51 immunoprecipitate rERG and two different Tara antibodies to detect Tara. An
82-85 kDa band detected by the Tara antibody also associated with rERG in 3 of
4 rats, and a band of ~55 kDa was detected in the immunoprecipitation lane, but was not seen in the input lysate. Although the immunoprecipitation and immunoblot antibodies were from different species, this ~55 kDa band may be immunoglobulin G (IgG) since it is present in high abundance. Tara’s association appeared to be specific for hERG since KCNQ1, a related potassium channel found in the heart, did not associate with Tara in rat heart (Fig. 3D).
Tara Localizes to M- and Z-lines.
To characterize Tara localization in rat ventricular myocytes, we used the
Tara antibody exhibiting the least non-specific binding on western blots. We co- stained with Myosin binding protein C (MyBPC) as a marker for sarcomeric organization (Fig. 4C). MyBPC is a thick filament-associated protein, which localizes to sarcomeric A-bands in a doublet pattern with the center space of each doublet defined as the M-line, and the space between each doublet the I- band, which contains the Z-line and T-tubules (Fig. 4A). Two signals were observed. The brighter signal (red) fell on the M-line border (Fig. 4D); longitudinal line profiles show the large peaks from the Tara antibodies (red) overlap with the MyBPC doublet (green) (Fig. 4D). A less intense signal appeared as puncta localized between each MyBPC doublet, consistent with Z- line/T-tubular location (Fig. 4D, arrows). Three dimensional reconstruction of an optically sectioned myocyte depicted Tara signal in large structures 52 corresponding to the M-line border (Fig. 4E and F, “M”) and smaller puncta along the Z-lines (Fig. 4E and F, arrows). These puncta correspond to previously published Z-line expression patterns for ERG (Jones et al., 2004).
Tara decreases hERG protein levels
Because the coimmunoprecipitation results suggested Tara interacts with hERG along the secretory pathway (Fig. 3B), we next determined whether Tara affects hERG expression and/or maturation. hERG immunoblots from HEK-293 cells over-expressing Tara did not consistently increase or decrease the mature fraction of hERG 1a signal (data not shown). However, overexpression of myc/his-tagged Tara decreased total hERG 1a expression, normalized to loading control, by 30% (+/- 8%) (N=5, p<.05) (Fig. 5A and B).
Overexpression of Tara decreased hERG expression, and to determine if a reduction in endogenous Tara levels would increase hERG expression, Tara was knocked down in HEK-293 cells. A 75% (+/-10%) (N=3, p<.05) reduction of endogenous Tara did not significantly alter hERG expression in HEK-293 expressing hERG 1a when compared to cells transfected with a non-targeting control (Fig. 5C and D).
Tara decreases hERG current density
To determine whether the decrease in hERG protein levels described above correlates to a functional decrease in hERG current amplitude, two-electrode voltage clamp recordings were taken from oocytes injected in a 1:3 molar ratio of 53 cRNA encoding hERG and Tara, respectively. Fully-activated tail current levels were measured, and a decrease of 39% (+/- 4%) (N=122 cells from 5 different animals, p<.05) was recorded in oocytes expressing hERG 1a and Tara compared to cells expressing hERG 1a alone (Fig. 6A). To verify Tara’s effect was specific and not due to down-regulation of hERG in order to synthesize overexpressed Tara, recordings from oocytes injected with hERG 1aΔC, were compared to oocytes expressing hERG 1aΔC and Tara. Tara had no effect on hERG 1aΔC currents (Fig. 6B), suggesting that hERG currents were suppressed by a mechanism involving the Tara-binding domain of the hERG C-terminus.
2.3. Discussion
This study demonstrates interaction of hERG with a novel binding partner,
Tara. Here, we show bands recognized by Tara antibodies migrate as a ~72 kDa band in human heart and HEK-293 cells, and as a 65-68 kDa band in dog and rat hearts. Tara associated with the mature and immature forms of hERG in HEK-
293 cells and coassociated with hERG in rat heart. In rat myocytes, Tara exhibited a periodic signal corresponding to the M-line, and a secondary signal corresponding to the Z-lines where ERG has been previously localized to T- tubules. Over-expression of Tara functioned to decrease hERG protein expression and current amplitude, suggesting it plays an important role in regulating hERG and possibly repolarization of the cardiac action potential.
Six isoforms of Tara, also known as Trio and F-actin binding protein
(TRIOBP) 1-6, have been identified with RT-PCR (Riazudden et al., 2006; 54 Shahin et al., 2006). While Tara, or TRIOBP-1, was detected by RT PCR and
Northern blot in all human tissue tested, including the heart and kidneys, TRIOBP
3-6 expression was limited to the retina, cochlea, and fetal brain (Seipel et al.
2001; Riazuddin et al. 2006; Shahin et al. 2006). The two anti-Tara antibodies used in this study were raised against epitopes common to Tara/TRIOBP-1, -3, -
5, and -6. None of the bands detected in heart tissue or HEK-293 cells corresponds to the predicted size of any other Tara/TRIOBP isoforms, which supports the RT PCR and Northern blot data that only Tara/TRIOBP-1 is expressed in human heart. These data also suggest that Tara/TRIOBP-1 is the only isoform in rat and dog hearts, and in HEK-293 cells.
While a consistent change in maturation was not detected in crude membrane preparations from HEK-293 cells over-expressing Tara, recent work suggests trafficking of hERG may be detected at the functional level while changes in hERG maturation go undetected in immunoblots of crude lysates
(Biliczki et al., 2009; Hayashi et al., 2010). Thus, decreases in the mature pool of
Tara may not have been detected. Due to variability in hERG maturation detected in whole cell lysates, a large sample size may be required to detect changes in hERG maturation. Using statistical power analysis to predict the number of samples needed to reject the null hypothesis without error, approximately 150 individual experiments are needed to achieve the standard
80% statistical power within the 5% confidence interval. While this number is quite large, modifying experimental techniques to analyze only surface localized hERG would likely reduce the error and the number of samples required to 55 achieve statistical significance.
Previous studies show a direct interaction of Tara with actin, and here we show Tara directly interacts with hERG in yeast two-hybrid assays. Cytoskeletal interacting proteins, such as filamin A and ankyrin, function in the heart and brain as scaffolding proteins to coordinate surface localization of ion channels and transporters (Mohler et al., 2003; Mohler et al., 2004). Tara may have a role in regulating surface localization of hERG through a mechanism other than trafficking. The Tara-mediated decrease in hERG protein expression may reflect a destabilization of channel complexes that function to recycle channels or target them for degradation.
Work by Yano et al. suggests a role for Tara in transcriptional regulation of
E-cadherin (Yano et al., 2011). While Tara functioned to up-regulate transcription of E-cadherin via a Rac 1-mediated pathway, it is possible that Tara may act to down-regulate hERG at the transcriptional level. Little is known about transcriptional regulation of hERG, and defining Tara’s role in down-regulation of hERG expression may provide important insight on the early events of hERG synthesis and regulation. Understanding these complexes may also provide important information about additional disease mechanisms.
Future studies are required to determine the mechanism by which Tara acts to decrease hERG expression. Tara decreases hERG expression and current amplitude and may play a critical role in channel trafficking and/or stability.
Understanding the relationship between hERG and Tara may provide novel information on hERG channel regulation. 56 2.4. Materials and Methods
Cell Membrane Protein Preparations-HEK-293 cells were maintained and transfected in 60 mm tissue culture treated dishes (Corning). Cells were washed with ice-cold PBS 48 hours post transfection and resuspended in lysis buffer (150 mM NaCl, 25 mM Tris-HCl, 5 mM glucose, 20 mM NaEDTA, 10 mM NaEGTA 10 mM, 1% Triton X-100, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56
µg/ml benzamidine, and 1x Complete Minitab (Roche Applied Science)). After sonicating twice (amplitude 20 for 10 seconds on ice), samples were rotated for
20 minutes at 4 °C, centrifuged at 10,000 x g at 4 °C, and supernatants analyzed.
Protein concentration determined using a modified Bradford assay (DC Protein
Assay, BIORAD)
Sprague-Dawley rat ventricles were excised from anesthetized adult males after injection of sodium pentobarbital (100 mg/kg body weight intraperitoneal) as described previously (He et al., 2001). Ventricular tissue was homogenized in tissue homogenization solution (25 mM Tris-HCl, pH 7.4, 10 mM NaEGTA, 20 mM NaEDTA, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche Applied Science)). After homogenization with tissue tearer (2 x 10 second bursts), lysates were sonicated twice at (amplitude 20 for 10 seconds on ice), and centrifuged at 1,000 x g for
10 minutes at 4°C. The supernatant was decanted and the pellet resuspended in tissue homogenization solution and the homogenization, sonication, and centrifugation repeated. Supernatants were combined and centrifuged at 40,000 x g for 30 minutes at 4°C, and pellets resuspended in RIPA buffer (150 mM NaCl, 57 50 mM Tris-HCl, pH 7.4, 1 mM NaEDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 µg/ml 1,10 phenanthroline, 0.7
µg/ml pepstatin A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche
Applied Science)), and incubated at 4°C, rotating, for 2-3 hours, and centrifuged at 10,000 x g for 10 minutes at 4°C to remove any insoluble material. The supernatants were retained for analysis. Integral membrane proteins from canine ventricular tissue (gift from Dr. Cynthia Carnes) were isolated as described for rat tissue.
Crude membrane preparations were prepared from human male and female ventricular tissue (gift from Dr. Andras Varro). Ventricular tissue was broken into small pieces in liquid nitrogen and homogenized Tris-EDTA buffer
(5mM Tris-HCl pH 7.4, 2mM EDTA, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche Applied
Science)) to a final concentration of 0.1g tissue/2.0 mls TE. Tissue was homogenized with tissue tearer, and homogenate centrifuged at 40,000 x g for
30 minutes at 4°C. The resulting pellet was solubilized in solublization buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,
0.1% sodium dodecyl sulfate, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin
A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche Applied Science)) to a final concentration of 0.1 g tissue/1 ml solublization buffer, and incubated for
2 hours at 4°C, rotating. Solublized proteins were then centrifuged at 4,000 x g for 10 minutes at 4°C, and the supernatant was analyzed. 58 Electrophysiology-Oocytes were isolated from anesthetized female frogs and treated with Liberase Blendzyme (Roche) according to protocols approved by the
University of Wisconsin Animal Use and Care Committee. All hERG constructs were cloned into a pGH19 vector, and Tara was cloned into pcDNA3-mycB/his
(Invitrogen). Purified cRNA (mMessage mMachine, Ambion) was quantified and injected using a Drummond calibrated oocyte injector. Oocytes were incubated at
18 °C for 48 hours. Recordings were carried out at room temperature with oocytes being stored at 18 °C in the dark while experiments were conducted.
Two-electrode voltage clamp experiments were carried out as described previously (Trudeau et al. 1995). Currents were recorded in a solution containing
5 mM KCl, 93 mM NaCl, 1 mMMgCl2, 0.3 mM CaCl2, and 5 mM HEPES, pH 7.4.
PClamp (Axon Instruments) and Origin 4.1 (Microcal Software, Inc.) were used for data analysis and plotting. Current amplitudes were measured as tail current peaks. All amplitudes are represented as averages ± S.E. A Student's t test was used to determine the significance of unpaired observations.
Immunoprecipitations-HEK-293 whole cell lysates (250 µg) were precleared with
30 µl protein A sepharose beads for 1 hour at 4 °C. After centrifugation (1 minute at 10,000 x g, 4 °C) to remove beads, 0.25 µg rabbit anti-hERG KA R2 was added to the supernatant and incubated for ~16 hours at 4 °C, rotating. Protein
Sepharose beads were then added and incubated for an additional 2 hours.
Immunoprecipitates were washed three times in 0.5 ml lysis buffer, and eluted 59 into 30 µl Laemmeli sample buffer (25 mM Tris-HCl, pH 6.8, 2% sodium dodecysulfate, 10% glycerol, 0.2 M DL-Dithiothreitol).
Integral membrane proteins isolated from rat, dog, and human ventricles
(3 mg) were precleared with 50 µl protein G sepharose beads for 1 hour. Beads were removed and 15 µl mouse anti-hERG antibody (Axxora) was added to the supernatant and incubated for ~16 hours at 4 °C, rotating. After incubation with
35 µl protein G sepharose beads for 2 hours, beads were washed 3x with 250 µl
(150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM NaEDTA, 0.1% Triton X-100, 50
µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine, and
1x Complete Minitab (Roche Applied Science)). Samples eluted in 30 µl
Laemmeli sample buffer (25 mM Tris-HCl, pH 6.8, 2% sodium dodecysulfate,
10% glycerol, 0.2 M DL-Dithiothreitol).
Immunocytochemistry-Experiments were done as previously described (Jones et al., 2004). Briefly, isolated myocytes were fixed in 2% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperatrue.
Myocytes were pre-blocked in phosphate buffered saline, pH 7.4, 0.1% Tween-
20, 5% BSA for 2 hours at 4°C, then incubated in diluted primary antibody overnight at 4°C with constant rotation. Antibodies were diluted in phosphate buffered saline, pH 7.4, 0.1% Tween- 20, 5% BSA: mouse anti-Tara.27 1:500, goat anti-hERG 1a N-20 (Santa Cruz) 1:10, rabbit anti- Tara 1:500, and rabbit anti-Myosin Binding Protein C 1:500. Myocytes were washed 3x for 1 hour in PBS, pH 7.4 + 0.1 % Tween-20. Cells were then incubated in secondary 60 antibody diluted in phosphate buffered saline, pH 7.4, 0.1% Tween-20, 5%
BSA, 10% serum for 2 hours at room temperature with rotation; all Alexa Flours
(Invitrogen-Molecular Probes) were diluted 1:1000 in antibody dilutent. Samples were washed 3X briefly, then 2X for 1 hour at 4°C in PBS, pH7.4 + 0.1%
Tween-20. Myocytes were viewed on a Bio-Rad MRC 1024 laser scanning confocal microscope, or Zeiss Axiovert 200 microscope with a 63X objective using optical sectioning as described in Roti Roti et al. and Jones et al., respectively.
Knockdown of Endogenous Tara-HEK-293 cells were maintained and transfected in 35 mm tissue culture treated dishes (Corning). HEK-293 cells were transfected with 6 ng Tara si-RNA (Dharmacon). Control experiments consisted of cells transfected with 6 ng of a non-targeting siRNA control (Dharmacon) in place of Tara siRNA. Cells were incubated 48-72 hours, lysed, and analyzed.
Transfection of HEK-293 cells-HEK-293 cells were plated in 60 mm tissue culture-treated dishes (corning) and incubated overnight at 37°C in 5% CO2 to achieve 60-80% confluency. Cells were transfected with 3 μg pcDNA3.1-Tara myc/his and/or 1 μg pcDNA3.1-hERG 1a using TransIT LT-1 (Mirus) following manufacturer’s conditions. Cells were incubated for 48 hours post-transfection and lysed.
Western Blots- HEK-293 whole cells lysates and integral membrane protein 61 preparations were separated by 7.5% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked and then probed with a 1:5000 dilution of rabbit anti-hERG KA, 1:500 dilution of rabbit anti-
Tara, or 1:250 dilution of rabbit anti-KCNQ1 (Sigma). Bands were imaged with chemiluminescence (Amersham ECL, GE Healthcare), or using the Li-Cor
Odyssey system. Statistically-significant differential expression was identified with Student t-tests (p<0.05 considered significant). Average data are shown as mean ± SEM.
Yeast Two-Hybrid Screen- Binary interactions were evaluated using the yeast 2- hybrid assay as previously described (Roti Roti et al., 2002). Briefly, PJ69-4a yeast were transformed singly or dually with plasmids containing recombinant clones fused to either the Gal4 Activation Domain (pACT2) or the Gal4 Binding
Domain (pAS2-1). Initial transformants were selected on synthetic dropout plates
(SD) lacking leucine, tryptophan, or both, as appropriate for the transformed vector(s). Colonies were replica-plated to selection media additionally lacking adenine or histidine; detecting growth on these plates indicates a protein-protein interaction. Yeast colonies were also replica-plated to selection media containing
X-Gal, where a blue colony phenotype indicates a positive protein- protein interaction. Representative colonies from each set of transformants were re- streaked onto SD-leu-trp and replica plated to interaction selection plates (-ade, - his, + X-Gal).
62 2.5. Authorship Note
Dr. Gail A. Robertson contributed to the study design and manuscript preparation.
Elon Roti Roti, with the assistance of Samantha Delfosse, conducted two-hybrid and colocalization studies in rat myocytes. Dr. Eugena M. C. Jones assisted with design of the Tara antigenic peptide used to make the rabbit anti-Tara antibody.
Dog ventricular tissue was a gift from Dr. Cynthia Carnes, and the human ventricular tissue a gift from Dr. Andras Varro.
63 2.6. Figures
Figure 1: Schematic of hERG and Tara proteins.
S1-S6 represent hERG transmembrane domains, and CNBD the cyclic nucleotide binding domain. The yellow oval denotes Tara’s pleckstrin homology domain.
Table 1: Summary of Yeast 2 hybrid data showing association of full-length and carboxy-terminus of Tara with the carboxy-terminus of hERG.
Growth of yeast (+) and blue reporter colonies indicate association of the hERG
C-termini and Tara, while no growth of yeast (-) and white reporter colonies indicate no association.
Figure 2: Endogenous Tara expression.
A. Immunoblots of isolated integral membrane proteins from human heart ventricles immunoblotted with rabbit anti-Tara (N=10). B. HEK-293 cells transfected with non-targeting (NT) or Tara-specific siRNA oligos and immunoblotted with the rabbit anti-Tara antibody. C. PDI immunoblots used as a loading control to determine 75% (+/- 10%) decrease of the 70-72 kDa Tara band (N=3, p< 0.05). D, E. Rat (D) and dog (E) integral membrane proteins immunoblotted with Mouse anti-Tara .27 (rat N=3, dog N=2) and rabbit anti-Tara
(rat N=4, dog N=9).
64 Table 2: Tara bands detected in human hearts.
Summary of all bands detected in human heart crude lysates immunoblotted with the rabbit anti-Tara antibody. Bands sizes given in kDa and N represents the number of times band was detected. Samples from 10 different donors were analyzed.
Table 3: Tara bands detected rat hearts.
Summary of all bands detected in integral protein preparations from rat hearts immunoblotted with the mouse anti-Tara .27 rabbit anti-Tara antibodies. Bands sizes given in kDa and the N is reported in parenthesis. Samples from 7 different rats were analyzed.
Table 4: Tara bands detected dog hearts.
Summary of all bands detected in integral protein preparations from dog hearts immunoblotted with the mouse anti-Tara .27 rabbit anti-Tara antibodies. Bands sizes given in kDa and the N is reported in parenthesis. Samples from 9 different dogs were analyzed.
Figure 3: ERG and Tara associate in a heterologous system and in rat ventricles.
A. Crude lysates from HEK-293 cells stably expressing hERG 1a co- immunoprecipitated with a mouse anti-hERG (A12) antibody and immunoblotted with rabbit anti-Tara antibody (N=6). B. Crude lysates from HEK-293 cells stably expressing hERG 1a co-immunoprecipitated with rabbit anti-Tara antibody and 65 immunoblotted with rabbit anti-hERG-KA (N=3). C. Integral membrane protein preparations from rat ventricles immunoprecipitated with mouse anti- hERG (A12) and immunblotted with rabbit anti-Tara antibody (N=4). D. Integral membrane protein preparations from rat ventricles immunoprecipitated with goat anti-KCNQ1 and immunblotted with rabbit anti-Tara antibody (N=2).
Figure 4: Tara localizes to M-and Z-lines.
A. Cartoon of sarcomere structure. B, C, and D. Rat myocytes stained with
Tara.27 (B) and MyBPC antibodies (C), and merged images (D). Arrows highlight punctate staining along Z-line (N=3 different animals, 15-20 cells per animal). Longitudinal line profiles for Tara (red) with MyBPC (green) were taken at multiple planes in optically sectioned myocytes. E. 3D image of Tara.27; M- line border marked M, Z-lines marked with arrows. F. Magnified image of 3E.
Figure 5: Tara decreases hERG expression
A. Lysates from HEK-293 cells expressing hERG 1a or hERG 1a and 3 µg Tara myc/his immunoblotted with rabbit anti-hERG-KA. Graph on right represents densitometry plotted as fold change in cells expressing hERG 1a and Tara compared to cells expressing hERG 1a alone. PDI used as a loading control
(N=5). B. Lysates from HEK-293 cells transiently expressing hERG 1a and Tara and immunoblotted for hERG, Tara, and PDI as a loading control (N=5, p<.05).
C. Lysates from HEK-293 stably expressing hERG 1a transfected with Tara siRNA oligos (Tara KD) or non-targeting (NT) siRNA, and immunoblotted with the 66 rabbit anti-hERG-KA and rabbit anti-Tara antibodies. PDI was used as a loading control. D. Fold change in hERG signal in cells transfected with non- targeting control compared to cells transfected with Tara-specific siRNA (N=3, p<.05).
Figure 6: Tara suppresses hERG currents in oocytes
A,B. Current-voltage plot of hERG tail currents recorded from Xenopus oocytes injected with hERG 1a (A) or hERG 1aΔC (B) cRNA in the presence or absence of Tara cRNA. C. Crude lysates from HEK-293 cells stably expressing hERG
1aΔC co-immunoprecipitated with a mouse anti-hERG (A12) antibody and immunoblotted with rabbit anti-Tara antibody (N=6).
Table 5. Summary of tail current amplitudes.
Tail current amplitudes from 5 different frogs. Condition denotes cRNA injected into cells, current is the average current recorded from the number of cells (n) in that experiment, and p represents probability, assuming the null hypothesis, of an unpaired student’s t test.
67 Figure 1.
Table 1: The Carboxy-terminus of hERG Directly Interacts with Tara
Bait Bait+Prey Bait+Prey Bait+Prey Bait Prey Selection Selection 1 Selection 2 Reporter -Trp(-Leu) -Trp(-Leu)-His -Trp(-Leu)-Ade -Trp(-Leu)+X-gal hERG C-term Tara C-term + + + Blue hERG C-term Tara + + + Blue Shaker C-term Tara C-term + - - White hERG C-term None + - - White hERG C-term Empty Vector + - - White hERG C-term pLAM5'-1 + - - White -Leu(-Trp) -Leu(-Trp)-His -Leu(-Trp)-Ade -Leu(-Trp)+X-gal Tara C-term hERG C-term + + + Blue Tara hERG C-term + + + Blue Tara C-term Shaker C-term + - - White
68 Figure 2.
A B
C
100 80
60
40 * 20 0 % Reduction in Tara % Reduction in Tara NT Tara KD
D E
69
Table 2: Bands detected by Tara immunoblot in human heart
Band Size (kDa) N 260 10 of 10 148‐150 10 of 10 145‐147 3 of 10 70‐73 10 of 10 68‐69 3 of 10 47 10 of 10
40 8 of 10
Table 3: Bands detected by Tara immunoblot in rat heart
Band Size (kDa) Mouse anti‐Tara .27 Rabbit anti‐Tara 82 X (4 of 4) 77 X (4 of 4)
65‐68 X (3 of 3) X (4 of 4)
49‐50 X (3 of 4) 46 X (4 of 4) 43 X (1 of 3) 39 X (1 of 3) 36 X (1 of 3)
Table 4: Bands detected by Tara immunoblot in dog heart
Band Size (kDa) Mouse anti‐Tara .27 Rabbit anti‐Tara 85 X (5 of 8) 65‐68 X (2 of 2) X (7 of 8) 60‐63 X (3 of 8) 58 X (6 of 8) 53 X (1 of 2)
48‐50 X (8 of 8)
70 Figure 3. M
A B
C D
71 Figure 4:
A
B C
D
M‐line Tara
M‐line doublet Z‐line Tara
72 E F
73 Figure 5.
A B
100 80 * 60 40 20 0 % Change in hERG 1a 1a 1a+Tara
C D
1.4 1.2 1 0.8
0.6
expression 0.4 Fold Change in hERG 0.2 0 NT Tara
74 Figure 6.
A
B
Table 5: Summary of current amplitudes from oocytes expression hERG with and without Tara
Frog Condition Current (µA) n p 1 1a 8.3 +/- 1.1 12 0.01 1a+Tara 4.1 +/- 0.9 9 2 1a 8.0 +/- 0.9 8 0.0002 1a+Tara 4.8 +/- 0.5 15 3 1a 3.5 +/- 0.3 20 0.01 1a+Tara 2.6 +/- 0.2 14 4 1a 4.9 +/- 0.8 10 0.0002 1a+Tara 3.2 +/- 0.6 13 1aΔC 5.8 +/- 0.5 16 0.3 1aΔC+Tara 5.1 +/- 0.6 20 5 1a 3.9 +/- 0.5 9 0.02 1a+Tara 2.3 +/- 0.4 12 1aΔC 6.3 +/- 0.5 15 0.75 1aΔC+Tara 6.2 +/- 0.6 13
75
2.7. References
Akhavan A, Atanasiu R, Noguchi T, Han W, Holder N, Shrier A (2005). Identification of the cyclic-nucleotide-binding domain as a conserved determinant of ion-channel cell-surface localization. J Cell Sci. 118(Pt 13):2803-12.
Anderson CL, Delisle BP, Anson BD, Kilby JA, Will ML, Tester DJ, Gong Q, Zhou Z, Ackerman MJ, January CT (2006). Most LQT2 mutations reduce Kv11.1 (hERG) current by a class 2 (trafficking-deficient) mechanism. Circulation. 113(3):365-73.
Biliczki P, Girmatsion Z, Brandes RP, Harenkamp S, Pitard B, Charpentier F, Hébert TE, Hohnloser SH, Baró I, Nattel S, Ehrlich JR (2009). Trafficking- deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction. Heart Rhythm. 6(12):1792- 801.
Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT (1995). A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80(5): 795-803.
Hayashi K, Shuai W, Sakamoto Y, Higashida H, Yamagishi M, Kupershmidt S (2010). Trafficking-competent KCNQ1 variably influences the function of HERG long QT alleles. Heart Rhythm. 7(7):973-80.
Jones EM, Roti Roti EC, Wang J, Delfosse SA, Robertson GA (2004). Cardiac IKr channels minimally comprise hERG 1a and 1b subunits. J Biol Chem. 279(43):44690-4.
Kitajiri S, Sakamoto T, Belyantseva IA, Goodyear RJ, Stepanyan R, Fujiwara I, Bird JE, Riazuddin S, Riazuddin S, Ahmed ZM, Hinshaw JE, Sellers J, Bartles JR, Hammer JA 3rd, Richardson GP, Griffith AJ, Frolenkov GI, Friedman TB (2010). Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing. Cell. 141(5):786-98.
Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogné K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, Bennett V (2003). Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature. 421(6923):634-9. 76
Mohler PJ, Splawski I, Napolitano C, Bottelli G, Sharpe L, Timothy K, Priori SG, Keating MT, Bennett V (2004). A cardiac arrhythmia syndrome caused by loss of ankyrin-B function. Proc Natl Acad Sci. 101(24):9137-42.
Papazian DM, Schwarz TL, Tempel BL, Jan YN, Jan LY (1987). Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science 237(4816):749-53.
Riazuddin S, Khan SN, Ahmed ZM, Ghosh M, Caution K, Nazli S, et al. (2006). Mutations in TRIOBP, Which Encodes a Putative Cytoskeletal-Organizing Protein, Are Associated with Nonsyndromic Recessive Deafness. Am J Hum Genet 78(1): 137-143.
Roti EC, Myers CD, Ayers RA, Boatman DE, Delfosse SA, Chan EK, Ackerman MJ, January CT, Robertson GA (2002). Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene. J Biol Chem. 277(49):47779-85.
Sanguinetti MC, Jiang C, Curran ME, Keating MT (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81(2): 299-307.
Seipel K, O'Brien SP, Iannotti E, Medley QG, Streuli M (2001). Tara, a novel F- actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization. J Cell Sci 114(Pt 2): 389-399.
Shahin H, Walsh T, Sobe T, Abu Sa'ed J, Abu Rayan A, Lynch ED, et al. (2006). Mutations in a novel isoform of TRIOBP that encodes a filamentous-actin binding protein are responsible for DFNB28 recessive nonsyndromic hearing loss. Am J Hum Genet 78(1): 144-152.
Tester DJ, Ackerman MJ (2009). Cardiomyopathic and channelopathic causes of sudden unexplained death in infants and children. Annu Rev Med 60: 69-84.
Trudeau MC, Warmke JW, Ganetzky B, Robertson GA (1995). HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269(5220): 92-95.
Yano T, Yamazaki Y, Adachi M, Okawa K, Fort P, Uji M, Tsukita S, Tsukita S (2011). Tara up-regulates E-cadherin transcription by binding to the Trio RhoGEF and inhibiting Rac signaling. J Cell Biol. 193(2):319-32.
Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA, January C (1998). 77 Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J. 74(1):230-41.
Chapter 3: Tara may facilitate association of hERG and KCNQ1
79
3.1. Introduction
Repolarization of the cardiac action potential is mediated by the K + delayed rectifier current, IK, which is composed of two distinct currents, the rapidly activating IKr and the more slowly activating IKs (Noble et al., 1969;
Sanguinetti et al., 1990). Disruption of IKr by decreased function or expression of channels encoded by the human ether-a-go-go related gene (hERG/KCNH2), and/or IKs by perturbations in KCNQ1 channels give rise to Long QT Syndrome
(LQTS), in which individuals experience episodes of cardiac excitability, syncope, torsade de pointe, and, in severe cases, sudden cardiac death (Curran et al.,
1995; Sanguinetti et al., 1995; Trudeau et al., 1995; Barhanin et al., 1996;
Sanguinetti et al., 1996). Mutations in KCNQ1 also cause Jervell Lange-Nielson syndrome in which afflicted individuals exhibit a prolonged QT interval and congenital deafness (Jervell et al., 1957; Neyroud et al., 1997).
When expressed independently hERG and KCNQ1 give rise to IKr and IKs, respectively. However, increasing evidence suggests these two channels interact.
Transgenic rabbits expressing hERG LQT2 pore mutants decrease KCNQ1 current and vice versa (Brunner et al., 2008). A physical link via their C-termini has been reported in CHO cells. However, the consequences of this interaction are controversial. Two separate groups report an increase in hERG current density when coexpressed with WT KCNQ1 (Biliczki et al., 2009; Hayashi et al.,
2010) and a separate group reported WT KCNQ1 decreased hERG current density and surface localization (Ren et al., 2010). Yeast two-hybrid experiments 80 in our laboratory show the C-termini of hERG and KCNQ1 do not directly interact, but both bind the C-terminus of “Trio associated repeat on actin” (Tara/TRIOBP-
1) (Roti Roti et al., 2002).
Tara is ubiquitously expressed and was first identified as a Trio binding protein that directly binds and stabilizes F-actin when expressed heterologously
(Seipel et al., 2001; Riazuddin et al., 2006; Shahin et al., 2006). Knock-out of
Tara is embryonic lethal, suggesting a critical role in development (Kitajiri et al.,
2010).
In this study, we tested the hypothesis that Tara mediates association of hERG and KCNQ1. Association of hERG and KCNQ1 in HEK-293 cells and canine ventricles support published reports showing interaction of these two channels. Over-expression of Tara increased association of hERG and KCNQ1 in HEK-293 cells, suggesting Tara may contribute to formation and/or functioning of the hERG/KCNQ1 complex.
3.2. Results
Confirmation of hERG and KCNQ1 association in a heterologous system and in canine ventricles.
Previous studies report an interaction of hERG and KCNQ1 in CHO cells
(Biliczki et al., 2009; Hayashi et al., 2010). To determine whether these channels associate in HEK-293 cells, lysates from cells overexpressing hERG 1a, 1b, and
KCNQ1 were immunoprecipitated for hERG and immunoblotted for KCNQ1. 81
Results show association of hERG with the 67-69 kDa KCNQ1 band (Fig. 1A).
As a control for hERG antibody specificity, coimmunoprecipitation experiments were repeated with lysates from cells expressing KCNQ1 alone. The hERG- specific antibody did not immunoprecipitate KCNQ1, verifying antibody specificity
(Fig. 1B). These results are the first to show association of hERG and KCNQ1 in
HEK-293 cells.
Recent studies show enhancement of hERG trafficking when coexpressed with KCNQ1 (Biliczki et al., 2009; Hayashi et al., 2010). hERG immunoblots of
HEK-293 lysates expressing hERG alone or hERG coexpressed with KCNQ1 showed no enhancement of hERG maturation or expression (1C and D).
To confirm the reported interaction of hERG and KCNQ1 in canine ventricles, integral membrane proteins from canine ventricular cardiac tissue were immunoprecipitated with a hERG antibody and immunoblotted for KCNQ1.
Results showed interaction of a 65-68 kDa band with the anti-hERG antibody
(Fig. 1E), which is smaller than the 75 kDa band reported to interact with hERG in canine heart (Ehrlich et al., 2004). While full-length KCNQ1 is predicted to migrate at 75 kDa, it has been shown to migrate between 64-75 kDa when overexpressed in heterologous systems (Ehrlich et al., 2004; Biliczki et al., 2009;
Hayashi et al., 2010; Ren et al., 2010).
KCNQ1 expression and association with hERG in human ventricles.
Studies of endogenous KCNQ1 in the human heart have relied on electrophysiological techniques with expression assayed at the transcript level. 82
We examined KCNQ1 protein expression in human ventricular tissue and detected a band at 67-69 kDa in 11 of 11 immunoblots with two different antibodies (Fig. 2A). One antibody also recognized bands at ~76 and 75 kDa in
2 of 2 immunoblots, which corresponds to the predicted size of 75 kDa (Fig. 2A).
It is possible that the rabbit anti-KCNQ1 antibody that detected only the 67-69 kDa band did not recognize the predicted 75 kDa band. Further consideration of signal sizes and antibody specificity is presented in the Discussion.
We next determined whether KCNQ1 and hERG associate in human heart ventricles. A 69 kDa band was detected with the KCNQ1 antibody in lysates immunoprecipitated for hERG, suggesting hERG and KCNQ1 associate in human ventricles (Fig. 2B).
The C-termini of hERG and KCNQ1 directly interact with the C-terminus of Tara
As described previously, our laboratory conducted a two-hybrid screen of a human heart cDNA library and discovered a direct interaction between the C- terminus of hERG and Tara. Using the same cDNA library, we found that the C- terminus of KCNQ1 also directly interacted with the C-terminus of Tara (amino acids 361-593) (Fig. 3 and Table 2). The interaction appeared specific to
KCNQ1 as the Tara C-terminal fragment failed to bind a related potassium channel found in Drosophila, Shaker B (Table 2) (Papazian et al. 1987).
Binary 2-hybrid tests confirmed the association of Tara’s C-terminal fragment with the C-terminus of KCNQ1. However, there was no direct association of the C-terminus of KCNQ1 with either full-length Tara or the C- 83 terminus of hERG (Table 2).
Tara associates with hERG but not KCNQ1 in a heterologous system
To determine if full-length Tara interacts with KCNQ1, HEK-293 cells overexpressing KCNQ1 and Tara myc/his were immunoprecipitated for KCNQ1 and immunoblotted for Tara. An interaction between Tara and KCNQ1 was not detected (Fig. 4A). This was not due to inadequate pull-down of KCNQ1 since
KCNQ1 immunoblots of the immunoprecipitated protein revealed efficient pull- down (Fig. 4B). Tara myc/his associated with hERG in HEK-293 cells, suggesting the myc/his tags did not inhibit association of Tara myc/his with
KCNQ1 (Fig. 4C). These results support data described in Chapter 2 suggesting hERG and Tara associate in a heterologous system, and show KCNQ1 and full- length Tara do not associate in HEK-293 cells, or that there is not enough of the
KCNQ1/Tara complex to detect by coimmunoprecipitation and immunoblot methods.
Overexpression of Tara increases association of hERG and KCNQ1
To test whether an increase in Tara affects association of hERG and
KCNQ1, HEK-293 cells overexpressing hERG, KCNQ1, and Tara were coimmunoprecipitated for hERG and immunoblotted for KCNQ1. A 3.1 fold (+/-
0.6) (N=3, p<0.5) increase in association of hERG 1a and KCNQ1 was detected in HEK-293 cells over-expressing Tara myc/his compared to cells expressing 84 hERG 1a and KCNQ1 alone (Fig. 5A and B), suggesting Tara is involved in the association of hERG and KCNQ1 complex.
Knockdown of endogenous Tara does not disrupt hERG-KCNQ1 association
Because overexpression of Tara resulted in enhanced association of hERG and KCNQ1, it follows that reduction of endogenous Tara should reduce association. We tested this hypothesis using siRNA knockdown in HEK-293 cells expressing hERG and KCNQ1. Although 90% knockdown of Tara was achieved, crude lysates immunoprecipitated with an anti-hERG antibody and immunoblotted for KCNQ1 showed no reduction in KCNQ1’s association with hERG (Fig. 6). The implications of these findings are discussed below.
3.3. Discussion
In this study, we provide data supporting published reports of association of hERG and KCNQ1 in canine ventricles and in a heterologous system, and show a 67-69 kDa band recognized by a KCNQ1 antibody interacts with hERG in human ventricles. Recent studies report a physical and functional interaction between hERG and KCNQ1, and our data suggests Tara may have a novel role in formation of the hERG/KCNQ1 complex. These findings may have important implications in understanding regulation of IKr.
Due to the difficulty of obtaining human heart tissue, few studies have investigated KCNQ1 at the protein level. Here, we identified ~150, 143, and 67-
69 kDa bands detected by KCNQ1 immunoblot in both dog and human ventricles. 85
In one study of canine ventricles, KCNQ1 is reported to migrate at 75 kDa, but it is unknown if the authors detected any additional bands since only a cut-out of the 75 kDa band was reported (Ehrlich et al., 2004). Two bands at 55 and 66 kDa have been identified by KCNQ1 immunoblot in rat heart with overexpression in heterologous systems ranging from 64-75 kDa (Jiang et al., 1997; Rasmussen et al., 2004, Brunner et al., 2008; Ren et al., 2009; Hayashi et al., 2010). There have been no previous reports of KCNQ1 in the ~143 and 150 kDa size range as we observed. These bands may represent aggregation of KCNQ1 protein, or non-specific binding of the antibody to abundant species in this size range. A splice variant with a truncated amino-terminus identified by RT PCR is predicted to migrate at 66 kDa, and has been shown to act as a dominant negative and inhibit full-length KCNQ1 current (Jiang et al., 1997). Here, the majority of human and canine immunoblots were probed with a rabbit anti-KCNQ1 antibody and detected a 67-69 kDa band. However, immunoblots with a different KCNQ1 antibody recognized bands ~75 and ~66 kDa, suggesting the rabbit anti-KCNQ1 may have recognized only the shorter isoform. The 67-69 kDa band may represent an abundant protein that is non-specifically recognized by the KCNQ1 antibodies. However, identification of this band with two KCNQ1 antibodies specific to different epitopes suggests the 67-69 kDa band represents KCNQ1.
While we cannot be certain whether the 67-69 kDa band represents full-length or truncated KCNQ1, our results show that this band interacts with hERG in human and canine heart. An interesting area of investigation is whether hERG associates with full-length and/or shorter KCNQ1 isoforms. Preferential 86 association of hERG with one isoform over the other may have profound consequences on repolarization of the cardiac action potential.
Although the C-terminus of Tara directly interacted with the C-termini of hERG and KCNQ1 in yeast two-hybrid studies, knockdown of endogenous Tara did not abolish interaction of hERG and KCNQ1 coexpressed in HEK-293 cells, suggesting Tara is not required for association of this complex. However, overexpression of Tara increased the association of hERG and KCNQ1 approximately 3 fold. Using a more sensitive technique, such as whole cell recordings from cells expressing hERG and/or KCNQ1 with and without Tara, may show functional changes in channels that were not detected by immunoprecipitation and immunoblot techniques (Biliczki et al., 2009; Hayashi et al., 2010). Immunocytochemistry of ventricular myocytes may also provide important information on whether hERG, KCNQ1, and Tara colocalize, and understanding where colocalization occurs could provide valuable insight into the function of the complex.
A role for KCNQ1 in trafficking of hERG channels has recently been reported (Biliczki et al., 2009; Hayashi et al., 2010). An increase in hERG current amplitude was recorded in CHO cells coexpressing wild-type KCNQ1 while coexpression of hERG and a trafficking-deficient KCNQ1 mutant either significantly decreased, or had no effect, on hERG current (Biliczki et al., 2009;
Hayashi et al., 2010). Biliczki et al. reported an increase in hERG surface localization and maturation when coexpressed with WT KCNQ1 while Hayashi et al. saw no change in hERG maturation. Here, we overexpressed hERG and 87 wild-type KCNQ1 and did not observe any increase in hERG maturation (Fig. 1C and D).
In Chapter 2 we showed Tara decreased hERG current amplitude and protein expression, and results of this study suggest involvement of Tara in formation of the hERG/KCNQ1 complex. One potentially unifying hypothesis is that Tara associates with hERG and KCNQ1 to mediate internalization. A recent study by Guo et al. (2011) shows 0 mM K+ conditions lead to degradation of hERG but not KCNQ1 when expressed individually, but when coexpressed, hERG and KCNQ1 are both degraded. Future experiments may reveal a novel role for Tara in regulation of the hERG/KCNQ1 complex.
3.4. Materials and Methods
Cardiac Tissue Preparation-Mongrel dogs were anesthetized by intravenous injection of pentobarbital sodium (dosage: 120 mg/kg for the first 4.5 kg and 60 mg/kg for every 4.5 kg thereafter). After anesthesia was achieved, the hearts were rapidly excised and perfused with cold cardioplegic solution (containing 5% glucose, 0.1% mannitol, 22.4 mM NaHCO3, and 30 mM KCl) injected into the coronary ostia. Left ventricular tissues were snap frozen in liquid nitrogen and stored at –80°C for protein analyses.
All procedures were approved by the Ohio State University Institutional
Animal Care and Use Committee and conformed with the Guide for the Care and 88
Use of Laboratory Animals published by the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996).
Cell Membrane Protein Preparations-HEK-293 cells were maintained and transfected in 60 mm tissue culture treated dishes (Corning). Cells were washed with ice-cold PBS 48 hours post transfection and resuspended in lysis buffer (150 mM NaCl, 25 mM Tris-HCl, 5 mM glucose, 20 mM NaEDTA, 10 mM NaEGTA 10 mM, 1% Triton X-100, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56
µg/ml benzamidine, and 1x Complete Minitab (Roche Applied Science)). After sonicating twice (amplitude 20 for 10 seconds on ice), samples were rotated for
20 minutes at 4 °C, centrifuged at 10,000 x g at 4 °C, and supernatants analyzed.
Protein concentration determined using a modified Bradford assay (DC Protein
Assay, BIORAD)
Canine ventricular tissue (gift from Cynthia Carnes) was homogenized in tissue homogenization solution (25 mM Tris-HCl, pH 7.4, 10 mM NaEGTA, 20 mM NaEDTA, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche Applied Science)). After homogenization with tissue tearer (2 x 10 second bursts), lysates were sonicated twice at (amplitue 20 for 10 seconds on ice), and centrifuged at 1,000 x g for 10 minutes at 4°C. The supernatant was decanted and the pellet resuspended in tissue homogenization solution and the homogenization, sonication, and centrifugation repeated. Supernatants were combined and centrifuged at 40,000 x g for 30 minutes at 4°C, and pellets resuspended in RIPA buffer (150 mM NaCl, 89
50 mM Tris-HCl, pH 7.4, 1 mM NaEDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 50 µg/ml 1,10 phenanthroline, 0.7
µg/ml pepstatin A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche
Applied Science)), and incubated at 4°C, rotating, for 2-3 hours, and centrifuged at 10,000 x g for 10 minutes at 4°C to remove any insoluble material. The supernatants were retained for analysis.
Immunoprecipitations-HEK-293 whole cell lysates (250 µg) were precleared with
30 µl protein A sepharose beads for 1 hour at 4 °C. After centrifugation (1 minute at 10,000 x g, 4 °C) to remove beads, 0.25 µg rabbit anti-hERG KA R2 was added to the supernatant and incubated for ~16 hours at 4 °C, rotating. Protein
A sepharose beads were then added and incubated for an additional 2 hours.
Immunoprecipitates were washed three times in 0.5 ml lysis buffer, and eluted into 30 µl Laemmeli sample buffer (25 mM Tris-HCl, pH 6.8, 2% sodium dodecysulfate, 10% glycerol, 0.2 M DL-Dithiothreitol).
Integral membrane proteins isolated from canine ventricles (3 mg) were precleared with 50 µl protein G sepharose beads for 1 hour. Beads were removed and 15 µl mouse anti-hERG antibody (Axxora) was added to the supernatant and incubated for ~16 hours at 4 °C, rotating. After incubation with
35 µl protein G sepharose beads for 2 hours, beads were washed 3x with 250 µl
(150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM NaEDTA, 0.1% Triton X-100, 50
µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine, and
1x Complete Minitab (Roche Applied Science)). Samples eluted in 30 µl 90
Laemmeli sample buffer (25 mM Tris-HCl, pH 6.8, 2% sodium dodecysulfate,
10% glycerol, 0.2 M DL-Dithiothreitol).
Knock-down of Endogenous Tara-HEK-293 cells were maintained and transfected in 35 mm tissue culture treated dishes (Corning). Cells were transfected with 1.25 µg pcDNA3-hERG 1a, 1.25 µg pcDNA3-hERG 1b, 0.5 µg pcDNA3-KCNQ1, and 6 ng Tara si-RNA (Dharmacon). Control experiments consisted of cells transfected with 6 ng of a non-targeting siRNA control
(Dharmacon) in place of Tara siRNA. Cells were incubated 48-72 hours, lysed, and analyzed.
Western Blots- HEK-293 whole cells lysates and integral membrane protein preparations were separated by 7.5% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked and then probed with a 1:250 dilution of rabbit anti-KCNQ1 (Sigma). Bands were imaged with chemiluminescence (Amersham ECL, GE Healthcare). Statistically- significant differential expression was identified with Student t-tests (p<0.05 considered significant). Average data are shown as mean ± SEM.
Yeast Two-Hybrid Screen- Binary interactions were evaluated using the yeast 2- hybrid assay as previously described (Roti Roti et al., 2002). Briefly, PJ69-4a yeast were transformed singly or dually with plasmids containing recombinant clones fused to either the Gal4 Activation Domain (pACT2) or the Gal4 Binding 91
Domain (pAS2-1). Initial transformants were selected on synthetic dropout plates
(SD) lacking leucine, tryptophan, or both, as appropriate for the transformed vector(s). Colonies were replica-plated to selection media additionally lacking adenine or histidine; detecting growth on these plates indicates a protein-protein interaction. Yeast colonies were also replica-plated to selection media containing
X-Gal, where a blue colony phenotype indicates a positive protein- protein interaction. Representative colonies from each set of transformants were re- streaked onto SD-leu-trp and replica plated to interaction selection plates (-ade, - his, + X-Gal) for generating figures.
3.5. Authorship Note
Dr. Gail A. Robertson contributed to the study design and manuscript preparation.
Elon Roti Roti, with the assistance of Samantha Delfosse, conducted 2-hybrid and colocalization studies in rat myocytes. Dog ventricular tissue was a gift from
Dr. Cynthia Carnes.
92
3.6. Figures
Figure 1: hERG and KCNQ1 associate in a heterologous system and in canine ventricles.
A, B. HEK-293 cells transiently transfected with hERG 1a, 1b, and KCNQ1 (A), or transfected with KCNQ1 and empty vector (B) were coimmunoprecipitated with an anti-hERG antibody and immunoblotted for KCNQ1. (N=4) C. hERG and KCNQ1 immunoblots of HEK-293 lysates of cells transiently expressing hERG 1a or hERG 1a+KCNQ1. Actin was used as a loading control. D.
Graphical representation of hERG % change expressed in the presence of absence of KCNQ1. (N=3) E. Isolated integral membrane proteins from mongrel canine ventricles were coimmunoprecipitated with an anti-hERG antibody and immunoblotted with an anti-KCNQ1 antibody. (N=3) IgG determined by presence of ~55 kDa band in only the immunoprecipitation lane.
Figure 2: KCNQ1 is expressed in human ventricles and associates with hERG.
A. Human heart ventricular lysate from the same donor immunoblotted with rabbit anti-KCNQ1 (N=11) or Goat anti-KCNQ1 (N=2). B. Human heart ventricular lysate immunoprecipitated with mouse anti-hERG (A12) and immunoblotted with rabbit anti-KCNQ1. (N=2)
Table 2: Immunoblot detection of KCNQ1 in human heart.
Summary of all bands detected by immunoblot using the rabbit anti-KCNQ1 93 antibody and goat anti-KCNQ1 antibody.
Figure 3: Schematic of KCNQ1 and Tara proteins.
Cartoon of KCNQ1 protein highlighting S1-S6 transmembrane domains, the A- kinase anchoring protein (AKAP), and the carboxy-terminal fragment of KCNQ1 used as bait. Tara cartoon highlights the peckstrin homology (PH) domain and carboxy-terminal coiled-coil domains and KCNQ1-binding region.
Table 2: Summary of Yeast 2 hybrid data showing association of the carboxy- terminus of Tara with the carboxy-terminus of KCNQ1.
Growth of yeast (+) and blue reporter colonies show association of the hERG C- termini and Tara, while no growth of yeast (-) and white reporter colonies indicate no association.
Figure 4: Tara associates with hERG but not KCNQ1 in a heterologous system.
A, B. Crude lysates from HEK-293 cells transiently transfected with KCNQ1 and
Tara myc/his coimmunoprecipitated with a KCNQ1 antibody and immunoblotted with a myc (A) or KCNQ1 (B ) antibody. (N=4) C. Crude lysates from HEK-293 cells stably expressing hERG 1a and 1b transiently transfected with full-length
Tara myc/his (middle panel) or empty vector (right panel), immunoprecipitated with rabbit anti-hERG KA antibody and immunoblotted with an anti-myc antibody.
(N=4). IgG determined by presence of ~55 kDa band in only the immunoprecipitation lane. 94
Figure 5: Overexpression of Tara increases association of hERG and KCNQ1.
A. Crude lysates from HEK-293 cells transiently expressing hERG 1a and
KCNQ1, or hERG 1a, KCNQ1, and Tara myc/his immunoprecipitated with the rabbit anti-hERG KA antibody and immunoblotted with for KCNQ1. Lysates were also immunoblotted for hERG, Tara, and PDI loading controls to quantify any changes in expression. (N=3, p<0.05) B. Graphical representation of 3.1 fold increase of KCNQ1 association of hERG when coexpressed with Tara.
Figure 6: Association of hERG and KCNQ1 in HEK-293 cells in which Tara has been knocked-down.
A,B. Knock-down of endogenous Tara in HEK-293 cells stably expressing hERG
1a and 1b using Tara-specific siRNA (A) or a scrambled oligo control (B). Crude lysates were co-immunoprecipitated using an anti-hERG antibody and immunoblotted for KCNQ1. Lysates were also immunoblotted for Tara and PDI
(loading control). 90% knock-down of Tara was achieved. (N=1) IgG determined by presence of ~55 kDa band in only the immunoprecipitation lane.
95
Figure 1.
IgG
C D
120
100 80 1a 60 1a+Q1 40 Expression 20
% Change in hERG 0
E
IgG
96
Figure 2.
A B
*
Table 1: Bands detected in human heart by KCNQ1 antibodies
Band Size Rabbit anti-KCNQ1 Goat anti-KCNQ1 150 X (1 of 13) X (1 of 2) 146 X (1 of 13) X (1 of 2) 130 X (1 of 2) 100 X (2 of 11) 94 X (1 of 11) 83 X (1 of 11) 76 X (2 of 2) 75 X (2 of 2) 73 X (1 of 2) 66 X (11 of 11) X (2 of 2) 56 X (2 of 11) X (1 of 2) 52 X (3 of 11) 42 X (2 of 11)
97
Figure 3.
Table 2.
Bait Bait+Prey Bait+Prey Bait Prey Selection Selection 1 Selection 2 Bait+Prey Reporter -Trp(-Leu) -Trp(-Leu)-His -Trp(-Leu)-Ade -Trp(-Leu)+X-gal KCNQ1 C- term Tara C-term + + + Blue KCNQ1 C- Tara full- term length + - - White KCNQ1 C- term hERG C-term + - - White KCNQ1 C- Empty term Vector + - - White Shaker C- term Tara + - - White
98
Figure 4.
IP: KCNQ1 Blot: Tara
IP: KCNQ1 Blot: KCNQ1
IgG
C
99
Figure 5.
A B
4
3 1a+Q1 2 1a+Q1+Tara 1 Fold Increase in KCNQ1 association 0
Figure 6.
IgG
100
3.7. References
Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M, Romey G (1996). K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature. 384(6604):78-80.
Biliczki P, Girmatsion Z, Brandes RP, Harenkamp S, Pitard B, Charpentier F, Hébert TE, Hohnloser SH, Baró I, Nattel S, Ehrlich JR (2009). Trafficking- deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction. Heart Rhythm. 6(12):1792- 801.
Brunner M, Peng X, Liu GX, Ren XQ, Ziv O, Choi BR, Mathur R, Hajjiri M, Odening KE, Steinberg E, Folco EJ, Pringa E, Centracchio J, Macharzina RR, Donahay T, Schofield L, Rana N, Kirk M, Mitchell GF, Poppas A, Zehender M, Koren G (2008). Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome. J Clin Invest. 118(6):2246-59.
Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT (1995). A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80(5): 795-803.
Ehrlich JR, Pourrier M, Weerapura M, Ethier N, Marmabachi AM, Hébert TE, Nattel S (2004). KvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currents. J Biol Chem. 279(2):1233-41.
Guo J, Wang T, Yang T, Xu J, Li W, Fridman MD, Fisher JT, Zhang S (2011). Interaction between the cardiac rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium channels revealed by low K+-induced hERG endocytic degradation. J Biol Chem. 286(40):34664-74.
Hayashi K, Shuai W, Sakamoto Y, Higashida H, Yamagishi M, Kupershmidt S (2010). Trafficking-competent KCNQ1 variably influences the function of HERG long QT alleles. Heart Rhythm. 7(7):973-80.
Jervell A, Lange-Nielsen F (1957). Congenital deaf-mutism, functional heart disease with prolongation of the Q-T interval and sudden death. Am Heart J. 54(1):59-68.
101
Jiang M, Tseng-Crank J, Tseng GN (1997). Suppression of slow delayed rectifier current by a truncated isoform of KvLQT1 cloned from normal human heart. J Biol Chem. 272(39):24109-12.
Kitajiri S, Sakamoto T, Belyantseva IA, Goodyear RJ, Stepanyan R, Fujiwara I, Bird JE, Riazuddin S, Riazuddin S, Ahmed ZM, Hinshaw JE, Sellers J, Bartles JR, Hammer JA 3rd, Richardson GP, Griffith AJ, Frolenkov GI, Friedman TB (2010). Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing. Cell. 141(5):786-98.
Neyroud N, Tesson F, Denjoy I, Leibovici M, Donger C, Barhanin J, Fauré S, Gary F, Coumel P, Petit C, Schwartz K, Guicheney P (1997). A novel mutation in the potassium channel gene KVLQT1 causes the Jervell and Lange-Nielsen cardioauditory syndrome. Nat Genet. 15(2):186-9.
Noble D, Tsien RW (1969). Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres. J Physiol 200(1): 205-231.
Papazian DM, Schwarz TL, Tempel BL, Jan YN, Jan LY (1987). Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science 237(4816):749-53.
Rasmussen HB, Møller M, Knaus HG, Jensen BS, Olesen SP, Jørgensen NK (2004). Subcellular localization of the delayed rectifier K(+) channels KCNQ1 and ERG1 in the rat heart. Am J Physiol Heart Circ Physiol. 286(4):H1300-9.
Ren XQ, Liu GX, Organ-Darling LE, Zheng R, Roder K, Jindal HK, Centracchio J, McDonald TV, Koren G (2010). Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines. Am J Physiol Heart Circ Physiol. 299(5):H1525-34. Riazuddin S, Khan SN, Ahmed ZM, Ghosh M, Caution K, Nazli S, et al. (2006). Mutations in TRIOBP, Which Encodes a Putative Cytoskeletal-Organizing Protein, Are Associated with Nonsyndromic Recessive Deafness. Am J Hum Genet 78(1): 137-143.
Roti EC, Myers CD, Ayers RA, Boatman DE, Delfosse SA, Chan EK, Ackerman MJ, January CT, Robertson GA (2002). Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene. J Biol Chem. 277(49):47779-85.
Sanguinetti MC, Jurkiewicz NK (1990). Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 96(1): 195-215.
102
Sanguinetti MC, Jiang C, Curran ME, Keating MT (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81(2): 299-307.
Seipel K, O'Brien SP, Iannotti E, Medley QG, Streuli M (2001). Tara, a novel F- actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization. J Cell Sci 114(Pt 2): 389-399.
Shahin H, Walsh T, Sobe T, Abu Sa'ed J, Abu Rayan A, Lynch ED, et al. (2006). Mutations in a novel isoform of TRIOBP that encodes a filamentous-actin binding protein are responsible for DFNB28 recessive nonsyndromic hearing loss. Am J Hum Genet 78(1): 144-152.
Trudeau MC, Warmke JW, Ganetzky B, Robertson GA (1995). HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269(5220): 92-95.
Yano T, Yamazaki Y, Adachi M, Okawa K, Fort P, Uji M, Tsukita S, Tsukita S (2011). Tara up-regulates E-cadherin transcription by binding to the Trio RhoGEF and inhibiting Rac signaling. J Cell Biol. 193(2):319-32.
103
Chapter 4: hERG Isoform Expression in Human Heart
104 4.1. Introduction
Channels encoded by the human ether-a-go-go-related gene (hERG) give rise to IKr current in the heart and determine action potential duration by contributing to the repolarization phase of the cardiac action potential
(Sanguinetti et al., 1995; Trudeau et al., 1995). Disruption of IKr by inherited mutations in hERG, or block of channels by pharmaceutical drugs, can cause the potentially fatal long QT Syndrome (Curran et al., 1995; Sanguinetti et al., 1995).
Most of our understanding of hERG comes from heterologous studies of the original hERG isolate, hERG 1a. However, increasing evidence suggests endogenous hERG channels are comprised of two isoforms, hERG 1a and hERG 1b. hERG 1a and 1b are identical except for alternate 5’ exons, and encode unique cytoplasmic amino-termini of significantly different sizes (Lees-
Miller et al., 1997; London et al., 1997). These two proteins associate in rat, dog, and human heart based on coimmunoprecipitation and immunocytochemical distributions (Jones et al., 2004). Expressed in HEK-293 cells and recorded at near physiological temperatures, the current evoked during an action potential voltage clamp command is nearly two times greater for hERG 1a/1b than hERG
1a channels (Sale et al., 2008). Thus, heteromeric 1a/1b currents provide more effective repolarization, a conclusion further supported by the identification of an
LQT2 mutation that reduces stability of the 1b protein (Sale et al., 2008).
Both hERG 1a and 1b transcripts have been detected by RT PCR in mouse, rat, guinea pig, dog, and human heart tissues, and 1a and 1b protein 105 immunodetected in mice, rats, dogs, and humans (Lees-Miller et al., 1997;
London et al., 1997; Zehelein et al., 2001; Jones et al., 2004; Wang et al., 2008).
Due to differences in cardiac depolarization/repolarization among mammalian species, animal studies do not necessarily predict torsade de pointe in humans, which highlights the importance of understanding hERG channel composition in humans. The limited studies investigating hERG isoform expression at the protein level in human tissue have been inconclusive.
This study attempts to fill this knowledge gap by evaluating expression of isoforms in human ventricular myocardium. Much of this report focuses on resolving technical issues that arise because of (1) limited access to undiseased tissue; (2) low abundance of ion channel proteins compared to total cellular protein and (3) sensitivity to proteases, making protein labile and difficult to isolate during long biochemical procedures.
4.2. Results
hERG expression in human heart
To examine hERG expression in human heart ventricles, membrane preparations were immunoblotted with a hERG antibody and recognized bands at 150-155 kDa and 135-138 kDa, which are consistent with previously published results identifying these bands as mature and immature hERG1a glycoforms, respectively (Fig. 1) (Jones et al., 2004; Gaborit et al., 2010). In addition, hERG 106 immunodetection revealed three lower molecular mass bands at 95-97 kDa,
83-86, and 80-83 kDa. The 95 and 85 kDa bands are consistent with hERG 1b expression in human heart and heterologous systems (Fig. 1A) (Jones et al.,
2004). The 80 kDa band is consistent with unglycosylated hERG, and glycosidase treatment of heterologously expressed 1b showed reduction of the
95 and 85 kDa to 80 kDa bands, suggesting unglycosylated hERG 1b migrates at
80 kDa (Jones et al., 2004). Detection of all bands is attributed to recognition of proteins by the hERG antibody since immunoblots using the only the secondary antibody were blank (Fig. 1B). We attempted to verify glycosylation states of mature, immature, and unglycosylated hERG 1a and 1b bands in human heart using glycosydases. Unfortunately, hERG was degraded after treatment and could not be detected by immunoblot.
hERG isoform expression in men and women
To determine if hERG subunit composition differs between the sexes, we compared 1a and 1b immunoblot signals in men and women. Of the total hERG signal, 1a accounted for 56% (+/- 13%, N=4) in men and 60% (+/- 12%, N=4) in women (Fig. 2). These values are not statistically significant, suggesting subunit composition does not vary by sex in humans. However, distributions more tightly associated with age groups may yet reveal important differences.
107 Expression of hERG 1a and 1b gycoforms in men and women
hERG 1a and 1b form tetrameric homomers when expressed alone in heterologous systems, but readily associate into heteromeric channels when coexpressed. The ratio of mature 1a to total hERG signal was calculated to be
11% (+/- 5%, N=4) in men and 14% (+/- 7%, N=4) in women and the ratio of mature 1b to total hERG was 28% (+/- 9%, N=4) in men and 26% (+/- 10%, N=4) in women (Fig. 3). As discussed below, these ratios are not statistically significant. Analysis of additional donors is required to reduce error and better understand hERG glycoform expression.
Homogenization of human ventricular tissue
To determine the optimal conditions for processing frozen human heart ventricular tissue into a liquid homogenate, tissue was either pulverized into a fine powder or broken into small pieces in liquid nitrogen using a mortar and pestle. The pulverized and broken tissue was placed in ice-cold TE with protease inhibitors and blended on ice with a chilled tissue tearer until no remaining pieces of tissue were visible. After completion of the entire solubilization protocol (Fig. 4), analysis of hERG showed breaking the tissue yielded enough hERG 1a and 1b to view by immunoblot, while pulverization resulted in inconsistent detection of hERG 1a and 1b subunits (Fig. 5). More time is required to pulverize tissue than to break it, which is performed in the absence of protease inhibitors thereby leaving proteins vulnerable to degradation by cellular proteases. 108 Ultrasonication is a technique used to disrupt cells, and to test whether ultrasonication of homogenates resulted in increased hERG yield, a homogenate was ultrasonicated on ice 2x at 1 watt on ice, or left untreated. In this experiment, ultrasonication decreased the amount of hERG 1b detected (Fig. 6). This experiment was conducted early in the trouble-shooting process, and hERG 1a signal often was very faint or was not detected. Heat generated during the ultrasonication process, and/or shearing of protein by the high frequency sound waves may have decreased the amount of hERG recovered.
Solubilization of hERG protein from human ventricular tissue
One of the most critical stages of integral protein isolation is extracting proteins from lipid membranes into an aqueous environment with the use of detergents. Here, we determined the optimal detergent concentration and solubilization buffer to tissue ratio for the solubilization of hERG protein. A protocol for solubilization of hERG from ventricular tissue had been in place in our laboratory, which used a combination of Triton X-100 (Tx-100), sodium deoxycholate, and sodium dodecyl sulfate (SDS) detergents. Although this protocol did not consistently isolate hERG from canine ventricles, and when it did gave very low yields, it provided a combination of detergents that could isolate hERG. To optimize the solubilization buffer detergent concentration, pellets obtained from centrifugation of the homogenates were incubated with solubilization buffer containing 1%, 2%, or 5% TX-100 for 2 hours at 4° C while keeping the concentration of sodium deoxycholate at 1% and SDS at 0.1%. Both 109 1% and 2% Tx-100 allowed for immunodetection of both hERG 1a and 1b, while 5% Tx-100 buffer yielded less hERG protein (Fig. 7). It is likely the high concentration of detergents in the 5% Tx-100 sample caused hERG to denature.
Subsequent experiments were carried out using solubilization buffer containing
1% Tx-100, 1% sodium deoxycholate, and 0.1% SDS.
In addition to determining the optimal detergent concentrations in the solubilization buffer, a detergent to sample ratio of 7.5 ml solubilization buffer for every gram of tissue was found to be critical for immunodetection of hERG. The detergent concentration and solubilization buffer to tissue ratio are both important in achieving the critical micelle concentration (CMC) for the detergents, which is essential for disruption of the lipid bilayer and micelle formation in solution.
Concentration of isolated hERG protein
Integral membrane proteins are not only difficult to isolate, but are also expressed in low abundance. To maximize the hERG signal detected by immunoblot, solubilized proteins were concentrated in microcon centrifugal filtration tubes (Millipore), which function to capture and concentrate proteins greater than 50 kDa. Concentrations of the final solubilized lysate examined included 1.5, 3, 6, 18 and 30x the volume loaded in the unconcentrated input lane. The 3x concentration, which measured 85 μg after concentration, generated the optimal hERG immunoblot signal (Fig. 8). No hERG 1a was detected in the 1.5x concentrated sample, suggesting that enough of the sample was lost, or degraded, during centrifugation, such that it could not be detected 110 (Fig. 8). Samples containing more than 3x the input contained too much protein and appeared as smears on the immunoblot so that hERG bands could no longer be identified (Fig. 8).
Storage and stability of hERG protein
To test how long hERG is stable after solubilization, ventricular lysate was subjected to SDS-PAGE and immunoblotted for hERG immediately following solubilization and at 5.5 hours post-solubilization. At 5.5 hours post-solubilization most hERG was degraded except for unglycosylated 1b (Fig. 9). This data suggests that any assays for hERG 1a and mature 1b must be completed in less than 5.5 hours post solubilization.
Healthy human heart tissue is a precious and rare resource, and to assess whether hERG could be preserved for future experiments, solubilized hERG lysates were frozen immediately after solubilization at -80°C. Four days after freezing, samples were thawed in ice and immunoblotted for hERG. Both hERG
1a and 1b were detected after thawing suggesting samples can be stored for future use (Fig. 10).
4.3. Discussion
In this study we established a protocol to isolate fragile hERG proteins from human heart tissue to document hERG expression in human tissue. We describe signals in immunoblots corresponding to hERG 1a and 1b. Here, we 111 show immunodetection of hERG bands at 150-155 kDa, 135-138 kDa, 95-97 kDa, 83-86, and 80-83 kDa consistent with previously published results identifying these bands as mature 1a, immature 1a, mature 1b, immature 1b, and unglycosylated 1b, respectively. Quantitative immunoblots showed no significant sex-dependent differences in 1a and 1b isoform or glycoform expression.
Since its discovery in 1995, hERG has been intensely studied. The difficulty in obtaining healthy human heart tissue and isolating these fragile proteins has resulted in only three published reports of hERG protein isolated from human heart (Pond et al., 2000; Jones et al., 2004; Gaborit et al., 2010).
Here, we detect 150-155 kDa and 135-138 kDa bands, which correspond to hERG 1a. Mature hERG 1a has been reported to migrate at ~155 and ~160 kDa and immature 1a at ~135 and ~140 kDa (Jones et al., 2004; Gaborit et al., 2010).
An additional report showed only one hERG band at 145 kDa (Pond et al., 2000).
This single band may represent immature 1a since it is more abundant than mature 1a (Fig. 3), thereby making it easier to detect even with low affinity antibodies. The size difference of immature hERG 1a reported in this study and by Pond et al. may be explained by variations in determining protein mobility.
We identified three bands corresponding to hERG 1b at 95-97 kDa, 83-86, and 80-83 kDa. While all three glycoforms of 1b have been detected in HEK-293 cells transiently expressing 1b, the only published report of hERG 1b expression in human tissue identified two bands at 94 and 83 kDa with two different antibodies (Jones et al., 2004). The immunoblot detection method used in this study utilized near IRdye infrared secondary antibodies, which generate sharper 112 bands than detection of chemiluminescence by film used in the Jones et al. study. It is possible that three 1b bands were present in the published study, but the chemiluminescence detection made the unglycosylated and immature 1b appear as a single band. Large amounts of immature and unglycosylated 1b can also appear as one band even when using the more sensitive IRdye detection method (compare Figs. 8 and 9).
Bands detected in this study correspond to published sizes of hERG 1a and 1b, and probing immunoblots with antibodies raised to different epitopes will help confirm their identity. Analysis of bands by mass spectrometry would also help confirm identity of hERG 1a and 1b in human heart.
Determining relative abundance of hERG 1a and 1b isoforms contributes to our understanding of subunit stoichiometry. In men 56% (+/- 13%) and in women 60% (+/- 12%) of total hERG signal is 1a, which provides preliminary evidence that hERG 1a and 1b may be expressed in a 2:2 ratio. Experiments to determine subunit stoichiometry of individual hERG channels are currently underway in our laboratory.
Recent work from our laboratory shows three antidepressants associated with a two- to three-fold higher risk for sudden cardiac death in women are more potent for 1a/1b than 1a (Abi-Gerges et al., 2011). It is well established that certain drugs are associated with higher sudden cardiac death in women.
However, there has been no evaluation of hERG subunit composition between the sexes. Results of this study show no significant difference in 1a and 1b expression in men and women, suggesting hERG subunit composition alone is 113 not responsible for differential drug block between the sexes. This study analyzed a relatively small sample size of 8 (4 men and 4 women). Variability of protein expression between individuals may account for differences in hERG 1a and 1b expression between the sexes not being statistically significant. Analyzing an additional ~35 donors would raise the statistical power of the study from 29% to 80% and may reveal statistically significant difference in isoform expression between the sexes. Although we did not detect differences in hERG 1a and 1b isoform expression between the sexes, a recent study by Wang et al. (2008), shows 1b expression is highest in neonatal mice and decreases with age. A potential area of study is whether or not 1b expression varies with age.
Analysis of mature and immature glycoforms show the majority of hERG signal is immature with mature 1a accounting for 11% (+/- 5%) of hERG signal in men and 14% (+/- 7%) in women. The trend is the same for 1b with mature 1b accounting for 28% (+/- 9%) of the signal in men and 26% (+/- 10%) in women.
The high abundance of immature glycoforms may be due to protection of channels in vesicles as the more through the ER and Golgi, and once targeted to the membrane they become more susceptible to proteolytic degradation.
Analysis of additional samples is required to reduce error and better understand expression of hERG glycoforms in the heart.
This study provides a foundation on which to build our understanding of native hERG subunit composition in the heart.
114 4.4. Materials and Methods
Isolation of hERG protein
Human male and female ventricular tissue was a gift from Dr. Andras
Varro. Ventricular tissue was broken into small pieces in liquid nitrogen and homogenized in Tris-EDTA buffer (TE) (5mM Tris-HCl pH 7.4, 2mM EDTA, 50
µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine
(freshly prepared), and 1x Complete Minitab (Roche Applied Science)) to a final concentration of 0.1g tissue/2.0 mls TE. Tissue was homogenized with tissue tearer (2 x 10 second bursts), and homogenate centrifuged at 40,000 x g for 10 minutes at 4°C. The resulting pellet was solubilized in solublization buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,
0.1% sodium dodecyl sulfate, 50 µg/ml 1,10 phenanthroline, 0.7 µg/ml pepstatin
A, 1.56 µg/ml benzamidine, and 1x Complete Minitab (Roche Applied Science)) to a final concentration of 1 g tissue/7.5 ml solublization buffer, and incubated for
2 hours at 4°C, rotating. Solubilized proteins were then centrifuged at 5,000 x g for 10 minutes at 4°C, and the supernatant analyzed.
Concentration of solubilized hERG
hERG lysates (~140 µg (1.5x), 240 µg (3x), 480 µg (6x), 1.4 mg (18x), and
2.4 mg (30x)) were diluted to a final volume of 500 µl with solubilization buffer without detergents (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 µg/ml 1,10 115 phenanthroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine, and 1x
Complete Minitab (Roche Applied Science)). Samples were then added to the sample reservoir of Microcon YM-50 tubes (Millipore) and centrifuged at 14,000 x g for 12 minutes at 4°C. After centrifugation, the sample reservoir was inverted into a clean 1.5 ml microfuge tube and centrifuged at 1,000 x g for 3 minutes at
4°C. The sample recovered in microfuge tube was analyzed.
SDS-PAGE and Immunoblots
hERG lysates were separated by 7.5% or 4-15% SDS-PAGE (BIORAD), and transferred onto polyvinylidene difluoride (PVDF-FL) (Millipore) membranes for 45 minutes at 4°C. Membranes were blocked in 5% milk diluted with phosphate-buffered saline containing Tween (PBS-T) for one hour at room temperature. Blocked membranes were then probed with mouse anti-GAPDH diluted 1:1000 and/or our pan rabbit anti-hERG KA R2 antibody diluted 1:2500 with block buffer overnight at 4°C. Membranes were washed in PBS-T for 45-60 minutes at room temperature, changing the wash buffer 3-4 times. Donkey anti- mouse IRdye 800 (LI-COR) and/or goat anti-rabbit IRdye 680 (LI-COR) were each diluted 1:15,000 in PBS-T and incubated with the membrane for one hour at room temperature. Membranes were washed as described above and imaged using the LI-COR Odyssey imaging system and software. Statistically-significant differential expression was identified with Student t-tests (p<0.05 considered significant). Average data are shown as mean ± SEM.
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4.5. Authorship Note
Dr. Gail A. Robertson contributed to the study design and manuscript preparation.
Sunita Joshi assisted with immunoblots comparing centrifugation times and speeds, and detergent concentrations. The human ventricular tissue was a gift from Dr. Andras Varro.
117 4.6. Figures
Figure 1: Immunodetection of bands corresponding to hERG 1a and 1b isoforms.
A. Isolated hERG from male and female donors was immunoblotted with the rabbit anti-hERG KA R2 antibody. Bands corresponding to mature and immature
1a, and mature, immature, and unglycosylated 1b were identified. B. Human ventricular lysate immunoblotted with only the secondary antibody.
Figure 2: No sex-dependent differences in hERG isoform expression.
Graphical representation of 1a signal/total hERG signal in male and female donors. Ratio calculated from quantitative rabbit anti-hERG KA R2 immunoblots of lysates from men and women normalized to GAPDH (N=8, 4 men and 4 women).
Figure 3: Comparison of hERG glycoforms in men and women.
Graphical representation of mature isoform signal/total hERG signal. Ratio calculated from quantitative rabbit anti-hERG KA R2 immunoblots of lysates from men and women normalized to GAPDH (N=8, 4 men and 4 women).
Red bars represent data from male donors and blue bars data from female donors. Solid bars represent hERG 1a and hatched bars hERG 1b.
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Figure 4: Summary of hERG isolation protocol.
Frozen human ventricular tissue was broken into small pieces in liquid nitrogen and homogenized using a tissue tearer in TE buffer containing protease inhibitors.
The homogenate was centrifuged at 40,000 x g for 10 minutes at 4°C, the resulting pellet incubated with solubilization buffer for 2 hours at 4°C, and then centrifuged at 5,000 x g for 10 minutes at 4°C. The supernatant was analyzed for hERG subunit expression.
Figure 5: Breaking of tissue yields more hERG protein than pulverization.
A, B. Frozen human ventricular was either pulverized (A) or broken into small pieces (B) in liquid nitrogen before being homogenized. After completion of the solubilization protocol, lysates were immunoblotted using the rabbit anti-hERG
KA R2 antibody. (N=2)
Figure 6: Sonication of human ventricular crude lysate does not increase hERG yield.
Homogenized human ventricular tissue was either incubated on ice (No sonication) or sonicated 2x at 1 watt for 20 seconds (Sonication) on ice before centrifugation. After completion of the solubilization protocol, protein was separated by SDS-PAGE and immunoblotted for hERG using the rabbit anti- hERG KA R2 antibody. (N=2)
119 Figure 7: Optimization of detergent concentration in solubilization buffer
Pellets obtained from homogenized human ventricular tissue were incubated with solubilization buffer containing 1%, 2%, and 5% Triton X-100. Solubilized proteins were separated by SDS-PAGE and immunoblotted with the rabbit anti- hERG KA R2 antibody. (N=1)
Figure 8: hERG protein can be concentrated for enhanced immunoblot signal.
Centricon tubes (Millipore) were used to concentrate ~140 µg (1.5x), 240 µg (3x),
480 µg (6x), 1.4 mg (18x), and 2.4 mg (30x) solubilized protein. All concentrated samples, including 80 µg of unconcentrated lysate (Input) were immunoblotted using the rabbit anti-hERG KA R2 antibody. (N=2)
Figure 9: Solubilized hERG protein cannot be detected by immunoblot 5.5 hours after isolation.
Solubilized hERG protein was separated by SDS-PAGE and immunoblotted for hERG immediately after isolation (0 min), or incubated on ice for 5.5 hours (5.5 hours) after isolation and then immunblotted using the rabbit anti-hERG KA R2 antibody. (N=1)
Figure 10: Solubilized hERG can be frozen for later use.
Solubilized hERG was either immunoblotted for hERG immediately after incubation with solubilization buffer (Fresh), or frozen at -80° C immediately after 120 solubilization and thawed 72 hours later (Frozen). All samples were separated by SDS-PAGE and immunoblotted with the rabbit anti-hERG KA R2 antibody.
(N=1)
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Figure 1:
A B
Mature 1a Immature 1a
Figure 2:
100
80
60 Male 40 Female
1a/Total hERG 1a/Total 20
0
122
Figure 3:
100
80 1a-Male
60 1a-Female
40 1b-Male 1b-Female 20 Mature/Total hERG Mature/Total
0
Figure 4:
Break tissue Homogenize Solubilize Solubilized in liquid tissue in TE buffer protein in hERG in nitrogen “homogenate” solubilization supernatant Centrifuge buffer Centrifuge at 40,000xg at 5,000xg
Figure 5.
A B
123 Figure 6.
Figure 7.
Figure 8.
124 Figure 9.
Figure 10.
125 4.7. References
Abi-Gerges N, Holkham H, Jones EM, Pollard CE, Valentin JP, Robertson GA (2011). hERG subunit composition determines differential drug sensitivity. Br J Pharmacol. 164(2b):419-32.
Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT (1995). A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80(5): 795-803.
Gaborit N, Varro A, Le Bouter S, Szuts V, Escande D, Nattel S, Demolombe S (2010). Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts. J Mol Cell Cardiol 49(4):639-46.
Jones EM, Roti Roti EC, Wang J, Delfosse SA, Robertson GA (2004). Cardiac IKr channels minimally comprise hERG 1a and 1b subunits. J Biol Chem. 279(43):44690-4.
Larsen AP, Olesen SP (2010). Differential expression of hERG1 channel isoforms reproduces properties of native I(Kr) and modulates cardiac action potential characteristics. PLoS One. 5(2):e9021.
Lees-Miller JP, Kondo C, Wang L, Duff HJ (1997). Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circulation Research 81(5):719-26.
London B, Trudeau MC, Newton KP, Beyer AK, Copeland NG, Gilbert DJ, Jenkins NA, Satler CA, Robertson GA (1997). Two isoforms of the mouse ether- a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circulation Research 81(5):870-8.
Phartiyal P, Jones EM, Robertson GA (2007). Heteromeric assembly of human ether-à-go-go-related gene (hERG) 1a/1b channels occurs cotranslationally via N-terminal interactions. J Biol Chem. 282(13):9874-82.
Pond AL, Scheve BK, Benedict AT, Petrecca K, Van Wagoner DR, Shrier A, Nerbonne JM (2000). Expression of distinct ERG proteins in rat, mouse, and human heart. Relation to functional I(Kr) channels. J Biol Chem 275(8):5997- 6006.
Sale H, Wang J, O'Hara TJ, Tester DJ, Phartiyal P, He JQ, Rudy Y, Ackerman MJ, Robertson GA (2008). Physiological properties of hERG 1a/1b heteromeric currents and a hERG 1b-specific mutation associated with Long-QT syndrome. Circ Res. 103(7):e81-95. 126
Sanguinetti MC, Jiang C, Curran ME, Keating MT (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81(2): 299-307.
Trudeau MC, Warmke JW, Ganetzky B, Robertson GA (1995). HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269(5220): 92-95.
Wang X, Xu R, Abernathey G, Taylor J, Alzghoul MB, Hannon K, Hockerman GH, Pond AL (2008). Kv11.1 channel subunit composition includes MinK and varies developmentally in mouse cardiac muscle. Dev Dyn. 237(9):2430-7.
Zehelein J, Zhang W, Koenen M, Graf M, Heinemann SH, Katus HA (2001). Molecular cloning and expression of cERG, the ether à go-go-related gene from canine myocardium. Pflugers Arch. 2001 May;442(2):188-91.
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Chapter 5: Conclusions and Future Directions
128
5.1 Summary
Proper functioning of hERG is critical in maintaining action potential duration and cardiac rhythmicity. Proteins that interact with hERG are increasingly being identified as regulators of action potential duration with disruption of these interactions giving rise to LQT syndromes. Here, we identified a novel hERG-interacting protein, Tara, by yeast two-hybrid studies, and provide evidence that hERG and Tara associate in heterologous and native systems.
Overexpression of Tara decreased hERG protein expression and current amplitude. In addition, coexpression of Tara with hERG and KCNQ1 increased association of hERG and KCNQ1 in HEK-293 cells, suggesting Tara may contribute to formation and/or functioning of the hERG/KCNQ1 complex. These studies contribute to our understanding of endogenous hERG-interacting proteins.
Due to the difficulty in obtaining healthy human heart tissue and the fragile nature of endogenously expressed hERG, few studies are carried out in human tissue. We established a protocol to isolate hERG proteins from human heart and identified bands 150-155 kDa, 135-138 kDa, 95-97 kDa, 83-86 kDa, and 80-
83 kDa, which correspond to mature 1a, immature 1a, mature 1b, immature 1b, and unglycosylated 1b, respectively. Analysis of isoform and glycoform expression in men and women showed no significant sex-dependent differences.
This study provides a foundation on which to build our understanding of native hERG channel subunit composition and may provide valuable insight into novel 129 cardiac repolarization regulatory mechanisms.
5.2 Future Directions
Pathways regulating decrease of hERG expression by Tara
In Chapter 2, we provide evidence that Tara interacts with hERG to decrease protein expression and current amplitude, which suggests Tara may regulate degradation of hERG. hERG degradation is regulated by proteasomal and lysosomal pathways (Gong et al., 2002; Akhavan et al., 2003; Chapman et al., 2005). To determine the if either of these pathways is involved in decrease of hERG expression by Tara, cells coexpressing hERG and Tara could be treated with proteasomal and lysosomal inhibitors. Abrogation of Tara’s decrease in hERG expression by an inhibitor would suggest a pathway in which Tara is involved. Subsequent studies may focus on regulation of the hERG/Tara complex by additional regulators of the identified pathway, which would provide valuable information about modulators of hERG degradation and potential disease mechanisms that arise from deregulation of these important pathways.
Effects of Tara mutants on hERG and action potential duration
Dr. Michael Ackerman, a collaborator at the Mayo Clinic, has identified
Tara mutants in a cohort of 400 LQT patients who tested negative for mutations in other known LQT loci. Seven mutants were identified, A91V, K130T, R271G,
R271Q, R341H, E474K, and G488S. Investigation into whether these mutants 130 give rise to a novel form of LQT is an interesting and exciting area of study.
Tara mutants may give rise to LQT by enhancing association of hERG or by acting as gain of function mutants, both of which would decrease hERG expression. Enhanced hERG degradation in myocytes would decrease the repolarizing current, and may give rise to LQT. To test the hypothesis that Tara mutant/s decrease hERG expression, immunocytochemistry, immunoprecipitation, and electrophysiology studies comparing cells expressing hERG and Tara to cells expressing hERG and Tara mutants, will show whether
Tara mutants enhance association with hERG, decrease hERG expression, and/or decrease current amplitude.
Role of Tara in association of hERG/KCNQ1 complex
hERG and KCNQ1, the two main channels responsible for repolarization of the cardiac action potential, have recently been shown to interact, with the effects of this association controversial. Whole cell voltage clamp recordings of hERG currents expressed alone and with KCNQ1 will help resolve discrepancies reported on the effects of KCNQ1 expression on hERG surface localization and current amplitude. In Chapter 2, we show Tara decreased hERG expression and current amplitude, and in Chapter 3, we show Tara over-expression increased association of hERG and KCNQ1. I hypothesize that the increased association of hERG and KCNQ1 mediated by Tara will decrease KCNQ1 expression. Whole cell recordings, immunoblot, and immunocytochemistry techniques in cells 131 expressing hERG and KCNQ1 in the presence and absence of over- expressed Tara will reveal affects of Tara on the hERG/KCNQ1 complex.
An additional area of study is whether Tara mutants disrupt, or enhance, association of hERG and KCNQ1. These studies may provide valuable information into regulation of hERG by KCNQ1 and potential disease mechanisms that arise from deregulation of the hERG/KCNQ1 complex.
5.3 Closing Remarks
Proper functioning of hERG is critical in determining action potential duration and maintaining cardiac rhythmicity. Surface localization of hERG is coordinated by multiple regulatory proteins, which we are just beginning to identify. Work in this thesis advances our knowledge of this field by elucidating a protocol to isolate hERG proteins from human heart tissue, which will contribute to studies investigating hERG subunit stoichiometry and native hERG protein complexes. In addition, we provide evidence of a novel interaction between hERG and Tara. Identification of Tara mutations in LQT patients who tested negative for mutations in other known LQT loci make Tara an exciting protein to study as a potential LQT target. The information gained from these experiments will be important in elucidating proteins involved in regulation of hERG channels and future studies may identify Tara as a novel LQT target.
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