CUI Zhi 201308 Master.Pdf (3.287Mb)

REGULATION OF THE HUMAN ETHER-À-GO-GO-RELATED GENE (HERG) CHANNEL BY RAB4 THROUGH NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWNREGULATED PROTEIN 4-2 (NEDD4-2)

ZHI CUI

A thesis submitted to the Physiology Graduate Program, Department of Biomedical and

Molecular Sciences

In conformity with the requirements for

the degree of Master of Science

Queen’s University

Kingston, Ontario, Canada

(August, 2013)

The human ether-à-go-go-related gene (hERG) encodes the pore-forming α-subunits of the Kv11.1 channel that is responsible for the cardiac rapidly activating delayed rectifier

+ K current (IKr), which plays a critical role in cardiac repolarization. Dysfunction of hERG causes long QT syndrome (LQTS), a cardiac electrical disorder that can lead to severe cardiac arrhythmias and sudden death (Mitcheson et al., 2000a; Roden, 2004;

Maier et al., 2006; Misner et al., 2012). The overall function of hERG channels is dependent on the channel density at the plasma membrane as well as proper channel gating. Previous work from our lab demonstrated that degradation of hERG protein in the lysosome is regulated by ubiquitin ligase Nedd4-2-mediated monoubiquitination (Sun et al., 2011; Guo et al., 2012). However, whether the internalized hERG proteins can be recycled back to the plasma membrane remains to be determined.

In the present study, we investigated the regulatory effects of various Rabs on hERG channels using Western blot analysis, co-immunoprecipitation (Co-IP), whole-cell patch clamp and immunofluorescence microscopy. The data revealed that, among hERG, human Kv1.5 (cardiac ultra-rapidly activating delayed rectifier K+ channel), and human

EAG (ether-à-go-go gene) potassium channels, Rab4 selectively decreased the mature hERG protein expression on the plasma membrane. Mechanistically, Rab4 did not directly target the internalized hERG protein for recycling. Instead, Rab4 increased the expression level of the E3 ubiquitin ligase Nedd4-2 (Neural Precursor Cell-expressed

Developmentally Downregulated Protein 4-2), which has been shown to mediate hERG ubiquitination and degradation (Guo et al., 2012). Nedd4-2 binding site mutations ∆1073

(binding site is removed) and Y1078A (binding site is modified) in hERG completely ii abolished the effect of Rab4. It has been shown that Nedd4-2 undergoes self- ubiquitination after targeting substrates (Bruce et al., 2008). My data further demonstrated that Rab4 decreased the degradation rate of Nedd4-2 and increased the rate of recycling. The increased Nedd4-2 then decreases hERG expression at the plasma membrane by targeting the PY-motif in the C-terminus of hERG channels.

In summary, the present study showed that Rab4 decreases the expression and function of hERG potassium channels on the plasma membrane through enhancing the recycling of the ubiquitin ligase Nedd4-2.

iii

Acknowledgements

First and foremost, I would like to thank my family: my mom, dad, aunts, and grandparents.

Thank you all for being so supportive of my studying abroad. I could not have accomplished such an achievement without the support from you all. Especially mom and dad, thank you for allowing me to come to Canada to make my dream come true. Mom had once told me that she wished that I could be around and see me every day, but she should not stop me from pursuing my dreams. She is everything any daughter could ever want. Without her help and support, I could not have seen such a big decision through. My dad is the kind of father that always cares and supports me so much, but does not show me the care directly. He is always behind me and guides me whenever I encounter difficulties. A mere “thank you” is not enough to express my gratitude for having such great parents. I would like to extend my appreciation to my fiancé,

Edwin, who got the journal started when he drove me all the way to Queen’s University for my interview, even after he worked a shift through the entire night. I appreciate his support over the course of my graduate study at Queen’s in all aspects. The expression “thank you” is not enough for me to express my feeling. I am so glad that I have such a great family.

Secondly, I would like to thank my supervisors Dr. Zhang and Dr. Fisher for all the support in the past two years. You are the greatest mentors a student could ever ask for. Your lessons not only provide my mind with much needed knowledge, but also trained my attitude towards research and improved my personal skills. Your encouragement meant so much to me during the past two years of my studies. I feel so blessed to have supervisors like you, who always put my priority first and foremost.

To my friends in the lab: Dr. Wang, Jun, Wentao, Tonghua, Yudi; you are all great people who are always willing to help me with my project. Shawn, Yan, Wonju, Andrew and Jeff: you are wonderful people that made the laboratory a fun place to stay. I owe my comfort and sanity in the lab each and every day to the warmth and kindness of you all – you kept me afloat even through iv the difficult experiments and unexpected results. The entire lab was like a family, which is something that not every lab can have, and for this I am so grateful.

Last, but not least, thank you to all my friends at Queen’s University and Toronto. Thank you all for being so supportive and accompanying me for the past two years on any occasion. It was a great time to be all together with you. Sally, my roommate, confidant, and partner in crime; and

Yudi, who was there with me through all those restless nights in the lab – I am so happy to have shared this time with you in the lab and in my life.

Table of Contents

Abstract ...... ii Acknowledgements ...... iv List of Figures ...... viii List of Abbreviations ...... x Chapter 1 Introduction and Literature Review ...... 1 1.1 The hERG Channel ...... 1 1.1.1 Regulation of hERG Channels ...... 3 1.1.2 The hERG Gating Properties ...... 6 1.2 Long QT Syndrome ...... 9 1.2.1 Inherited Long QT Syndrome ...... 11 1.2.2 Acquired Long QT Syndrome...... 12 1.3 Small GTPase-binding Protein ...... 14 1.3.1 Background of Small GTPases ...... 14 1.3.2 Function of Rab4 GTPase ...... 15 1.3.3 Alteration of Rab GTPase Expression and Related Diseases ...... 17 1.4 Ubiquitin-Proteasome System ...... 19 1.4.1 Ubiquitination ...... 19 1.4.2 Cascade of Ubiquitination ...... 21 1.4.3 Ubiquitin Ligase E3 ...... 23 1.4.3.1 Nedd4-1 ...... 23 1.4.3.2 Nedd4-2 ...... 24 1.4.3.3 Regulation of Nedd4-1 and Nedd4-2 ...... 26 1.5 Hypothesis ...... 29 1.6 Objective ...... 30 Chapter 2 Materials and Methods ...... 31 2.1 Molecular Biology and Cell Transfection ...... 31 2.2 Neonatal Rat Ventricular Myocyte Isolation ...... 32 2.3 Western Blot Analysis ...... 33 2.4 Co-Immunoprecipitation (Co-IP) ...... 33 2.5 Immunofluorescence Microscopy ...... 34 2.6 Electrophysiological Recording ...... 35 2.7 Reagents and Antibodies ...... 37

2.8 Statistical Analysis ...... 38 Chapter 3 Results ...... 39 3.1 Overexpression of Rab4 Decreases the hERG Expression Level at the Plasma membrane 39

3.2 Overexpression of Rab4 Decreases the hERG Current (IhERG) ...... 39 3.3 The effect of Rab4 is Specific to hERG Potassium Channels ...... 42 3.4 Knockdown of Rab4 Expression Increases hERG Protein Expression...... 42 3.5 Rab4 Increases hERG Ubiquitination ...... 42 3.6 The Interaction Between hERG and Nedd4-2 is Increased by Rab4 Overexpression ...... 45 3.7 Disrupting the hERG-Nedd4-2 Interaction Eliminates the Effect of Rab4 on hERG Channels ...... 45 3.8 Overexpression of Rab4 Increases the Endogenous Nedd4-2 Expression Level...... 49 3.9 Rab4 Increases Nedd4-2 Expression by Affecting the Nedd4-2 Degradation Rate ...... 49 3.10 Rab4 Impedes the Degradation Rate of Nedd4-2 ...... 52 3.11 Catalytic Activity of Nedd4-2 is Necessary for Rab4-mediated Enhancement of Nedd4-2 ...... 55 3.12 Rab4 Specifically Interacts with Catalytically Active Nedd4-2 ...... 55 3.13 Rab4 Decreases the Ubiquitination of Nedd4-2 ...... 57 3.14 Effect of Rab4 on the Expression of the ERG ChannelS in Neonatal Rat Cardiomyocytes ...... 60

3.15 Effect of Rab4 on the Function of IKr in Neonatal Rat Cardiomyocytes...... 60 3.16 Immunofluorescence Microscopy Revealing the Effects of Rab4 on the Expression of Both the ERG Channel and Nedd4-2 in Neonatal Rat Cardiomyocytes ...... 63 Chapter 4 Discussion ...... 65 Future Directions ...... 71

vii

List of Figures

Figure 1: Structure of the hERG Potassium Channel ...... 2 Figure 2: Scheme of hERG Gating Properties ...... 7

Figure 3: IKr Dysfunction Induces a Prolonged QT Interval and Abnormal Cardiac Action Potential ...... 10 Figure 4: Recycling of Internalized Plasma Membrane Proteins is Regulated by Rab GTPase ... 16 Figure 5: Ubiquitination ...... 22 Figure 6: Scheme Depicting the Regulation of Nedd4-2 Self-ubiquitination ...... 27 Figure 7: The Protocol for Whole-cell Patch Clamp Current Recording...... 36 Figure 8: Rab4 Decreases the Expression Level of Mature hERG Protein on the Plasma membrane ...... 40

Figure 9: hERG current (IhERG) is Decreased by Rab4 Overexpression ...... 41 Figure 10: Overexpression of Rab4 Neither Affects the Expression nor the Function of Kv1.5 and EAG channels ...... 43 Figure 11: Knockdown of Endogenous Rab4 Increases the Expression of hERG Channels in hERG-HEK Cells ...... 44 Figure 12: Rab4 enhances hERG ubiquitination ...... 45 Figure 13: Rab4 Increases the Interaction between Nedd4-2 and the hERG Channel ...... 47 Figure 14: Nedd4-2 is Involved in the Rab4-mediated Reduction of Mature hERG Channels .... 48 Figure 15: Effect of Overexpressed Rab4 on Endogenous Nedd4-2 Expression ...... 50 Figure 16: Confocal Image Showing Rab4 Increases the Endogenous Expression of Nedd4-2. .. 51 Figure 17: Inhibition of Nedd4-2 Degradation Eliminates the Rab4-mediated Increase in Nedd4-2 Expression ...... 53 Figure 18: Rab4 Interferes with the Degradation Process to Increase Nedd4-2 Expression ...... 54 Figure 19: The Rab4-mediated Enhancement of Nedd4-2 is Dependent on the Catalytic Activity of Nedd4-2 ...... 56 Figure 20: Rab4 Interacts with Catalytically Active Nedd4-2 ...... 58 Figure 21: Rab4 Decreases the Ubiquitination of Nedd4-2...... 59 Figure 22: Effect of Rab4 on the Expression of ERG in Neonatal Rat Cardiomyocytes...... 61

Figure 23: Effect of Rab4 on IKr in Neonatal Rat Cardiomyocytes ...... 62 Figure 24: Effect of Rab4 on the Expression of ERG and Nedd4-2 in Neonatal Rat Cardiomyocytes ...... 64

viii

Figure 25: A Proposed Scheme Depicting How Rab4 Increases the Nedd4-2 Expression Level by Facilitating Nedd4-2 Recycling ...... 67

List of Abbreviations

ALLN N-acetyl-leu-leu-norleucinal

BSA Bovine Serum Albumin

Ca2+ Calcium Ion cAMP Cyclic Adenosine Monophosphate

CHX Cycloheximide

DUBs Deubiquitinating Enzymes

E6AP human papilloma virus E6 Associated Protein

EAG Ether-à-go-go Gene

ECG Electrocardiography

ENaC Epithelial Sodium Channel

ER Endoplasmic Reticulum

ERG Ether-à-go-go Related Gene

FBS Fetal Bovine Serum

GDI GDP Dissociation Inhibitor

GEF Guanine nucleotide Exchange Factor

GGT Geranylgeranyl Transferase

GI Gastrointestinal

GS2 Griscelli Syndrome Type 2

HECT Homologous to E6-AP-COOH Terminus

HEK Human Embryonic Kidney hERG human Ether-à-go-go Related Gene

IKr Rapidly Activating Delayed Rectifier Potassium Current

IKs Slowly Activating Delayed Rectifier Potassium Current

K+ Potassium Ion x

Kv Voltage-gated potassium channel

LDL Low Density Lipoprotein

LQTS Long QT Syndrome

MEM Minimum Essential Medium

MG132 N-CBZ-leu-leu-leucinal

MJD Machadojoseph disease protease mV Millivolts

Na+ Sodium Ion

Nedd4-2 Neural Precursor Cell-expressed Developmentally Downregulated protein 4-2

OUT Otubain protease

PBS Phosphate- Buffered Saline

PBS-2 Phosphate- Buffered Saline supplemented with glucose

PCR Polymerase Chain Reaction

PVDF Polyvinylidene Difluoride

REP Rab Escort Protein

RING Really Interesting New Gene

RIPA Radioimmunoprecipitation

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis

SGK Serum/Glucocorticoid regulated Kinase siRNA small interfering RNA

TBS Tris-Buffered Saline

TSH Thyroid Stimulating Hormone

Ub Ubiquitin

UCH Ubiquitin C-terminal Hydrolase

USP Ubiquitin-Specific Protease

WT Wild Type

xii

Chapter 1

Introduction and Literature Review

1.1 The hERG Channel

Potassium channels play an important role in various physiological events, such as maintaining resting membrane potential (Hodgkin & Huxley, 1947), participating in cell metabolism (Craig et al., 2008) and regulating heart rhythm (Sanguinetti & Tristani-Firouzi,

2006). Human ERG (ether-à-go-go related gene) channels are widely expressed in tissues such as the inner ear, heart, smooth muscle in the gastrointestinal (GI) tract, and tumor cells (Warmke &

Ganetzky, 1994; Bianchi et al., 1998; Wang et al., 2002; Farrelly et al., 2003; Nie et al., 2005). hERG encodes the pore-forming -subunits of Kv11.1 channels that conduct the rapidly activating delayed rectifier current (IKr) in the heart, which plays a critical role in the plateau and repolarization phase of cardiac action potentials (Sanguinetti et al., 1995). The plateau phase ensures sufficient Ca2+ influx for optimum excitation of the ventricular myocytes. Reduced function of hERG channels, induced by either genetic defects or drug block, will cause long QT syndrome (LQTS), which is characterized by a prolonged QT interval on an electrocardiography

(ECG).

hERG channels share a similar structure with other tetramer-type voltage-gated K+ channels

(Kv), and are also composed of four identical subunits, each containing six transmembrane domains (S1-S6), as well as a large cytoplasmic NH2 terminus and a COOH terminus

(Sanguinetti & Tristani-Firouzi, 2006; Vandenberg et al., 2012) (Figure 1). While the S1-S4 domain comprises the voltage-sensing component, S5 and S6 with the large P loop together form

Pore

S S S S S S 1 2 3 4 5 6

C-terminus N-terminus

Figure 1: Structure of the hERG Potassium Channel. Representation of an α-subunit of the hERG channel. Each α-subunit is composed of six transmembrane domains (S1-S6) with a large cytoplasmic NH2 terminus and COOH terminus. S4 acts as a voltage sensor with seven positive charges. S5 and S6 form the pore.

the pore, which contributes to the selectivity filter of the channel. The identical α-subunits form a permeable aqueous pore in the center. The outer one third of the pore (extracellular end) lined by the large pore P loop that forms a narrow cylinder; and the inner two-thirds of the pore

(intracellular end) lined by the carboxyl-halves of the S6 domain that forms a water filled region called the central cavity (Doyle et al., 1998; Mitcheson et al., 2000a; Sanguinetti & Tristani-

Firouzi, 2006). The GFG sequence in the pore region of hERG channels determines the permeability of K+ (Snyders, 1999).

1.1.1 Regulation of hERG Channels The ERG (ether-à-go-go related gene) channel is a member of the EAG (ether-à-go-go) channel family and is widely expressed in a variety of species, such as rats, mice, guinea pigs, as well as humans (within the atrium and ventricle) (Sanguinetti & Jurkiewicz, 1991; Wang et al.,

1994; Wang et al., 1996; Wymore et al., 1997; Pond & Nerbonne, 2001; Zhang, 2006; Guo et al.,

2007). The ERG channel on Western blot analysis displays two distinct bands and the molecular masses of the two bands slightly vary depending on the species. Within cardiac myocytes, IKr conducted by ERG channels is an important K+ currents that mediate repolarization during the cardiac action potential; whereas within non-cardiac cells, ERG channels participate mainly in the maintenance of the resting membrane potential (Schwarz & Bauer, 2004).

The exogenous expression of human ERG (hERG) channels in the HEK 293 stable cell line, analyzed by Western blotting, revealed that hERG channel protein displays two forms. The immature form of hERG, primarily localized in the endoplasmic reticulum (ER), is a core- glycosylated protein with a molecular mass of 135 kDa. The immature hERG is subsequently transported to the Golgi apparatus for full glycosylation with a molecular mass of 155 kDa, which is eventually inserted in the plasma membrane. Only mature hERG channels are stable on the

plasma membrane as functional channels and are responsible for conducting the K+ current. The application of N-glycosidase F, an amidase that catalyzes the hydrolysis of asparagine-linked high mannose from glycoproteins, reduces the molecular masses of both bands to lower levels, further indicating that both forms of hERG protein exist in a glycosylated form (Zhou et al.,

1998b).

Glycosylation is critical for the maturation and stability of hERG protein. Two extracellular consensus sites for N-linked glycosylation were found in hERG protein. Initially, Petrecca et al.

(1999) showed that the presence of both extracellular N-linked glycosylation sites (N598, N629) are required for the transition of hERG from the immature to the mature form, as well as the proper expression of hERG on the plasma membrane. Later, Gong et al. (2002) specified that the

N629 mutation causes a hERG trafficking deficiency and leads to its intracellular retention, whereas N598 is essential for N-linked glycosylation but is not required for channel surface expression. Thus, not only correctly folded protein, but also proper trafficking and high stabilization on the plasma membrane are required for functional hERG channels.

The expression of hERG protein is also regulated by several other factors, including low extracellular K+ concentration, ubiquitin ligase Nedd4-2, serum/glucocorticoid regulated kinase

(SGK) and temperature (Vandenberg et al., 2006; Guo et al., 2009; Massaeli et al., 2010; Albesa et al., 2011; Guo et al., 2012; Lamothe & Zhang, 2013). Roden and Hoffman (1985) suggested that hypokalemia (reduced extracellular K+ concentration) can prolong the cardiac action potential, which indicated the role of K+ in the normal function of cardiac action potential.

Recently, our lab showed that extracellular K+ plays a role in stabilizing hERG channels on the plasma membrane, as low extracellular K+ concentration decreases the mature hERG protein

expression by accelerating its internalization and degradation (Guo et al., 2009; Massaeli et al.,

2010).

In addition, the ubiquitin ligase, Nedd4-2, has been shown to play an important role in the degradation of the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) (Debonneville et al., 2001; Malik et al., 2005; Rotin & Staub,

2012). Albesa et al. (2011) and Guo et al. (2012) also showed that Nedd4-2 is involved in ubiquitination and degradation of hERG channels. Nedd4-2 recognizes the PY motif of the mature hERG channels on the plasma membrane and labels them with a single ubiquitin molecule. Moreover, caveolin 3, a signature protein of caveolae, is also involved in Nedd4-2 mediated hERG degradation by recruiting Nedd4-2 to the cell membrane (Guo et al., 2012).

SGK has also been shown to participate in the regulation of hERG expression via the phosphorylation of Nedd4-2 (Lamothe & Zhang, 2013). A recent publication from our lab showed that SGK1 and SGK3 can up-regulate hERG function and expression by decreasing the activity of Nedd4-2 via phosphorylation. Moreover, SGK3 can up-regulate the function of Rab11, which indirectly increases the recycling of internalized hERG protein (Lamothe & Zhang, 2013).

These results extend our understanding of the mechanism of SGK3-mediated hERG function and expression.

Temperature also affects the expression and function of hERG channels. Chen et al. (2007) showed that low temperature can help the maintenance of hERG protein on the cell membrane and rescue some trafficking-deficient hERG mutants, while Vandenberg et al. (2006) demonstrated that high temperature altered the gating properties of hERG channels by shifting the voltage-dependent activation in a hyperpolarizing direction. The mechanisms of temperature- induced alteration of hERG channel function require further investigation.

1.1.2 The hERG Gating Properties

hERG is a member of the ERG family and shares a similar structure with other Kv channels, but unlike other Kv potassium channels, hERG channels possess some unique gating properties

(Figure 2). It is well known that in the voltage-gated K+ channels the S4 domain, which contains positively charged lysine or arginine residues in every third position, functions as the primary voltage sensor (Sanguinetti & Tristani-Firouzi, 2006). The S4 domain moves outward during the depolarization phase and returns to its original position when the membrane potential repolarizes.

Besides the common characteristics of potassium channel characteristics, hERG channels possesses two unique properties: (1) inactivation is faster than activation; and (2) recovery from inactivation is faster than that of deactivation (Sanguinetti & Tristani-Firouzi, 2006). The detailed hERG gating properties can be described in the following manner: when the membrane potential is held at -80mV, hERG channels are closed and no K+ current is detected. Upon depolarizing, the number of hERG channels that are open increases as the membrane potential becomes more positive. Channels are maximally open at a voltage of 20mV, where the biggest hERG tail current is detected. As the membrane potential becomes more positive, the opened hERG channels quickly enter the inactive state, which limits the amount of outward K+ current. As membrane potential becomes more negative during the late repolarization stage, hERG channels recover from inactivation within a few milliseconds and reopen, leading to a big outward current.

Recovered hERG channels quickly enter a closed state, where any current eventually disappears

(Schönherr & Heinemann, 1996; Smith et al., 1996; Zhou et al., 1998b; Perry et al., 2010;

Vandenberg et al., 2012) (Figure 2).

Applying this to the cardiac action potential, phase 2 (plateau phase) is maintained because

A Depolarization Repolarization K+ Outer slow fast

Inner Closed Open Inactivation

Recovery pA 2000B Deactivation 1500

1000

Open Inactivation Y Axis Title Axis Y 500 Closed

0 2000 4000 6000 8000 10000 12000 X Axis Title

Figure 2: Scheme of hERG Gating Properties. A, gating properties of the hERG channel.

hERG channel cycles between closed, open, and inactive states. B, hERG current trace. The

hERG channel opens in a voltage dependent manner. It rapidly enters inactivation after opening.

A repolarizing step to -50mV can recover all inactivated channels followed by channel

deactivation. The tail current during recovery is used for data analysis.

inactivation of hERG channels occurs quicker than activation during the early repolarization stage. During the late stage of repolarization, the quick recovery of hERG channels from inactivation increases the outward IhERG and initiates phase 3 (fast repolarization), returning membrane potential.

The mechanisms of the voltage sensor S4 have been extensively studied in Kv channels.

Despite their similar structures, the charge distribution in the voltage sensing domains between hERG and other Kv channels differs. The hERG channel has three additional negative charges in its voltage-sensing S2 and S3 domains (-2, -4, and -5) compared to the charges possessed by other

Kv channels. In addition, hERG possesses seven positive charges in the S4 domain, one less than that of other Kv channel family members (Tseng, 2001; Zhang et al., 2004). In terms of function, it is the first four positive charges in the S4 domain of the Kv channels that sense transmembrane voltages during activation and change their positions. The negative charges in the voltage sensing domains serve to stabilize the positive charges within S4 by forming salt bridges, which further stabilize the channel during open or closed states.

With regard to the inactivation of hERG channels mentioned previously, it has been proposed that it is the lack of H bonds at the outer mouth that makes hERG channels easily enter inactivation compared with other Kv channels (Zhang et al., 2004). In addition, the linker between S5 and S6, which is longer in hERG channels than in other Kv channels, can cause a conformational change during activation by affecting the function of the outer mouth of the channel and induce fast inactivation of hERG channel (Tseng, 2001; Zhang et al., 2004). Besides the natural structure-determined rapid inactivation, the detection of Na+ current through hERG channels during inactivation in the absence of extracellular K+ reveals that hERG inactivation is composed of two phases: an initial rapid unstable state and a slow more stable phase (Gang &

Zhang, 2006). Mutations that removed the rapid inactivation of hERG channels abolished the Na+ current, suggesting that sodium ions flow through the rapid inactivation phase. Moreover, extracellular Na+ also demonstrated a potentially inhibitory effect on hERG channels (Mullins et al., 2002). Mutations at the pore helix and the S6 domain abolished the inhibitory effect of Na+ on the hERG channel, suggesting that the interaction site with Na+ is mainly at the outer pore of the channel.

1.2 Long QT Syndrome

Long QT syndrome (LQTS) is a type of cardiac disorder, characterized by a prolonged QT interval reflected in the electrocardiogram (ECG) (Curran et al., 1995). The first identified LQTS case was reported in 1957 by Jervell and Lange-Nielsen in a deaf-mute child (Jervell & Lange-

Nielsen, 1957). Later on, Ward (1964) also reported similar symptoms in non-deaf-mute patients.

LQTS affects 1 in every 5,000 to 10,000 people worldwide and predisposes affected individuals to a high risk of ventricular arrhythmias (Torsade de pointes) and sudden death (Moss et al.,

1991; Curran et al., 1995; Sanguinetti & Tristani-Firouzi, 2006). Alterations of ionic currents that contribute to the cardiac action potential underlie this pathological condition (Figure 3).

As a standard, LQTS is diagnosed on an ECG as a QT interval above 450ms in males and

470ms in females (Schwartz & Crotti, 2011). Given that IKr contributes a significant portion to the normal repolarization of cardiac action potentials, abnormal function of hERG channels conducting IKr will lead to delayed repolarization and prolonged QT interval on an ECG. Two forms of LQTS exist: (1) the inherited form due to genetic defects, and (2) the acquired form primarily as a result of drug blockade (Curran et al., 1995; Trudeau et al., 1995; Snyders &

Chaudhary, 1996).

A 50 50

IKr IKr

0 0

-50 -50

-100 -100 200 ms 200 ms

B R R

P T P T

Q Q S S

QT interval QT interval

Figure 3: IKr Dysfunction Induces a Prolonged QT Interval and Abnormal Cardiac Action

Potential. A: model showing a normal cardiac action potential (left) and hERG channel dysfunction induced prolongation of cardiac repolarization (right). B: normal ECG trace (left) and abnormal ECG with prolonged QT interval (right). P wave indicates atrium depolarization; QRS complex and T wave represent ventricular depolarization and repolarization, respectively. QT interval is the time span between ventricular depolarization and repolarization.

1.2.1 Inherited Long QT Syndrome

Inherited LQTS, also known as familial LQTS, is a relatively rare congenital heart disease.

LQTS type 1 (LQT-1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the -subunits of the slowly activating delayed rectifier potassium channel (IKs) (Russell et al., 1996; Moss et al., 2007). LQTS type 2 (LQT-2) is caused by hERG mutations, which encodes the pore-forming α-subunits of the rapidly activating delayed rectifier current (IKr). These two disorders account for the majority of inherited LQTS. In LQT-2 patients, approximately 450 loss-of-function hERG mutations have been identified, and most of them are missense mutations which can cause subunit misfolding, assembly issue, or trafficking deficiencies (Tanaka et al.,

1997; Satler et al., 1998; Sanguinetti, 2010).

The most common mutation sites of hERG are around the transmembrane domains and the pore helix. To better investigate the mechanisms of inherited LQTS, cell lines that stably express hERG mutations, either at the pore helix or at the membrane-spanning domains, were generated to study the abnormal function and expression of hERG mutations in vitro. For example, some missense mutations (V612L, T613M, and L615V) located at the pore helix region of hERG channel subunits induced little or even no current (Huang et al., 2001). In addition, Zhou et al.

(1998a) also showed that hERG channel mutations at Y611H and V822M exhibited abnormal channel processing, The T474I mutation altered gating properties , and I593R and G628S mutations generated non-functional channels. Other mutations, such as hERG G601S, located at the extracellular region between S5 and the pore, induces a protein trafficking – deficiency, leading to no hERG expression on the plasma membrane (Akimoto et al., 1998). Such results demonstrate that pore helix and transmembrane domains are important for normal channel

expression and function. These mutations mimic hERG mutations in vivo and provide directions for potential pharmaceutical therapies.

To date, not all abnormal functions of hERG channels have been identified. The relationship between mutation sites and mutation-induced dysfunction requires further investigation, and strategies to rescue mutant hERG channels are essential for correcting LQTS. Mutant hERG channels are degraded along the ubiquitin-proteasome pathway (Gong et al., 2005). Alteration of the degradation pathway may be a potential strategy to rescue the function of hERG mutants.

1.2.2 Acquired Long QT Syndrome

Acquired LQTS is more common than inherited LQTS. It is mostly caused by drug-induced blockage of hERG channels (Roden et al., 1996; Karle et al., 2001). In the past decade, prolongation of QT interval, as an adverse effect of some drugs, called for the withdrawal of these drugs from the market. Multiple studies have shown that drugs, such as antihistamines, antibiotics, anti-arrhythmic and antipsychotic agents can affect the function of hERG channels and reduce K+ current (Roden, 1998, 2004). The high sensitivity of hERG channels to so many different medications presents a major hurdle for pharmaceutical companies that must evaluate the effect of each drug on hERG channel function in order to prevent this undesirable side effect.

Other known predisposing factors to LQTS include hypokalemia, congestive heart failure, and gender with females more susceptible than males (Roden, 2004).

In theory, drugs may affect any type of ion channel in the heart that contributes to the cardiac action potential. However, due to the high sensitivity of hERG channels to drugs, attention has been drawn to investigate the relationships and mechanisms of drug-induced hERG channel blockage. Two possibilities have been proposed: firstly, drugs that bind directly to a specific region of the channel pore can decrease its permeability to K+ (Lees-Miller et al., 2000; 12

Mitcheson et al., 2000a). These drug binding sites are primarily located within the pore helix and the transmembrane domains (Lees-Miller et al., 2000; Perry et al., 2010). Second, trapping of charged organic compounds, such as anesthetics, in the inner cavity that then block the activation gate of the channel (Mitcheson et al., 2000b). Phe652 and Tyr656, which are hydrophobic residues in the S6 transmembrane domain, are unique to ERG voltage-gated K+ channel and responsible for the high affinity of drug to the channel (Sanguinetti & Mitcheson, 2005).

Mutations in either of these two amino acid residues decrease the drug sensitivity of the hERG channel (Ficker et al., 1998; Lees-Miller et al., 2000). In addition, Perry et al. (2010) showed that amino acids on the S6 transmembrane domain (G648, Y652, and F656) and the pore helix

(T623 and V652), which are unique to EAG/ERG K+ channels, are the binding sites for antiarrhythmic drugs. Secondly, drugs such as probucol, fluoxetine and norfluoxetine, can potentially interfere with the trafficking of hERG protein and ultimately the expression level on the plasma membrane (Maier et al., 2006; Guo et al., 2007). For example, fluoxetine and norfluoxetine not only directly block the hERG channel but also interfere with hERG protein trafficking (Maier et al., 2006). Because the overall function of hERG channels depends on the number of functional channels on the plasma membrane, either reduction of channel expression or a disruption of channel gating properties will significantly impair the channel function and normal cardiac rhythm.

Given the complexity of the multiple drug binding sites within hERG channels and the mechanisms of drug-induced dysfunction of hERG channels, it is crucial to design pharmacological interventions that can not only avoid this severe side effect, but can also rescue hERG channel mutations (Robertson & January, 2006).

1.3 Small GTPase-binding Protein

1.3.1 Background of Small GTPases

Small GTPases are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP). These G-proteins are monomeric proteins with consensus amino acid domains that can switch between binding with guanosine diphosphatase and guanosine triphosphatase depending on their activity (Pochynyuk et al., 2007). More than 100 small

GTPases have been discovered in eukaryotes, of which the Ras GTPase superfamily is one of the better known groups. Ras GTPase is classified into 5 subfamilies: Rab, Sar1/Arf, Ras, Rho, and

Ran. Each member plays a distinct role within cells. Rab is involved in regulating vesicle formation and trafficking between intracellular compartments and the plasma membrane (Gurkan et al., 2005); Sar1 regulates the trafficking of proteins from the early endosome to the Golgi apparatus (Kuge et al., 1994); Ras is involved in regulating cell growth, differentiation and apoptosis (Olson & Marais, 2000); Rho regulates both cytoskeleton reorganization and gene expression (Ridley, 1997); and Ran functions at the level of regulating nucleocytoplasmic transport during the G1, S, and G2 phases (Moore, 1998).

Rab GTPases have been shown to play an important role in maintaining the steady-state of proteins at the cell surface by regulating formation, trafficking and fusion of vesicles (Stenmark

& Olkkone, 2001; Stenmark, 2009). More than 60 Rab GTPases have been identified in mammalian cells, but only a select few have been well studied (Jordens et al., 2005; Grosshans et al., 2006). It has been shown that Rab GTPases are primarily located at the cytoplasmic face of intracellular compartments, and different Rabs are found in distinct intracellular compartments with specific functions in regulating protein trafficking (Stenmark & Olkkone, 2001). The specificity of Rab GTPase localization is determined by residues within the C-terminus (Chavrier 14

et al., 1991). Rab1 and Rab2 are located at the ER and the Golgi complex, and are involved in regulating protein transport from the ER to the Golgi (Dupré et al., 2006). Rab4 is localized at the early sorting endosome, and is responsible for the rapid and direct recycling from the early endosome to the plasma surface (Daro et al., 1996; Mohrmann & Sluijs, 1999). Rab5, also located at the early endosome, regulates the retrograde endocytic trafficking from the plasma membrane to the early endosome and the subsequent transport to the recycling endosomal compartment (Bucci et al., 1992). Rab6 is required for intra-Golgi transport (Dupré et al., 2006).

Rab7 and Rab9 are identified as late endosomal markers, regulating vesicle transport to and from the late endosome (Chavrier et al., 1990; Lombardi et al., 1993). Rab11 is a recycling endosomal marker mediating slow recycling from the recycling endosome to the plasma membrane (Ullrich et al., 1996).

1.3.2 Function of Rab4 GTPase

Rab GTPases are involved in multiple intracellular signaling pathways, such as vesicle budding, uncoating, motility and fusion (Zerial & McBride, 2001; Grosshans et al., 2006;

Stenmark, 2009). As mentioned previously, Rab4 is located at the early endosome and regulates the rapid recycling of proteins from the early endosome to the plasma membrane (Figure 4).

Since Rab4 regulates the recycling of proteins at the early endosome, the expression of the recycled protein should theoretically be increased. Potokar et al. (2012) demonstrated that Rab4 and Rab5 are critical for regulating the directional motility of vesicles. The GDP-bound (inactive)

Rab4 and Rab5 significantly decrease the motility of vesicles and induce enlarged early endosomes in astrocytes. Moreover, Liu et al. (Liu et al., 2010) suggested that SGK can increase the function of Rab4 and facilitate the rapid recycling of synaptic α-Amino-3-hydroxy-5-methyl-

4-isoxazolepropionic acid (AMPA) receptors to the plasma membrane. In addition, Rab4 has 15

Figure 4: Recycling of Internalized Plasma Membrane Proteins is Regulated by Rab

GTPase. The homeostasis of proteins at the plasma membrane is a balance between anterograde trafficking and retrograde trafficking (degradation). After proteins experience internalization, a portion of the proteins can be recycled back to the plasma membrane by either Rab4 at the early endosome, or by Rab11 at the recycling endosome. Those proteins that are not recycled are targeted for degradation in the lysosome.

been shown to mediate the rapid recycling of Angiotensin II type 1 receptor (AT1R) and the beta-

2 adrenergic receptor via a cargo-mediated mechanism (Yudowski et al., 2009; Esseltine et al.,

2011).

Aside from mediating up-regulation of protein via the rapid recycling pathway, Rab4 is also involved in the down-regulation of protein expression levels. Saxena et al. (2006a; 2006b) showed that Rab4 reduced the expression and function of CFTR and ENaC on the plasma membrane. These results are contrary to the proposed function of Rab4-mediated rapid recycling of endocytic vesicles from the early endosome back to the plasma membrane. If the expression of

ENaC and CFTR is regulated by Rab4 via its proposed function, Rab4 should, in theory, facilitate the recycling of internalized ENaC and CFTR, leading to an enhanced expression level on the plasma membrane. However, these findings suggested that Rab4 may play a role not only in sorting CFTR and ENaC from early endosome to the plasma membrane, but also promoting them along the degradation route.

In summary, Rab GTPases participate in multiple cellular signaling events, facilitating the transportation of molecules between and within compartments and the plasma membrane

(McCaffrey et al., 2001; Jordens et al., 2005). Specifically, Rab4 mediates the rapid recycling of proteins at the early endosome, leading to an altered protein expression and function.

1.3.3 Alteration of Rab GTPase Expression and Related Diseases

Given that Rab GTPases play significant roles in the regulation of vesicle trafficking among and between intracellular compartments and the plasma membrane, genetic mutations or altered function (up- and down-regulated) of Rab GTPases have been commonly associated with diseases, such as cardiomyopathy (Wu et al., 2001), prostate cancer (Calvo et al., 2002), thyroid adenoma (Croizet-Berger et al., 2002), and lung tumor progression (Yao et al., 2002). The first 17

disease linked to a Rab mutation was Griscelli Syndrome Type 2 (GS2), an autosomal recessive disorder characterized by immune impairment and increased susceptibility to infection (Griscelli et al., 1978). The defective Rab27a in GS2 fails to induce secretary lysosome release from immune cells and platelets, resulting in the bleeding disorder and immune dysfunction in GS2

(Stinchcombe et al., 2001).

Specific to the function and expression of Rab GTPases in cardiovascular diseases, a reciprocal effect has been identified between the altered expression level of Rab GTPases and their related diseases. In 2001, Wu et al. (2001) discovered that the expression of Rab GTPases, such as Rab1, Rab4 and Rab6, were augmented in a dilated cardiomyopathy model expressing the

β2-adrenergic receptors (β2ARs). They further investigated the role of Rab4a in cardiomyocytes using transgenic mice, and found that overexpression of Rab4a in mice hearts can also induce cardiac hypertrophy, which progresses to heart failure in a time- and dose-dependent manner.

Structural analysis of cardiomyocytes revealed increased transitional vesicles and enlarged Golgi stacks. In addition, Rab4-mediated rapid recycling of β-adrenergic receptors has been found in both cell lines stably expressing β2ARs and in cardiomyocytes. The inactive Rab4-N121I mutant can block the recycling of β2ARs from the early endosome back to the plasma membrane in the cell line overexpressing β2ARs, but dephosphorylation of β2ARs was unaffected (Seachrist et al.,

2000). Consistent with the exogenously expressed β2AR, Filipeanu et al. (2006) suggested that

WT Rab4 regulates the recycling of β-adrenergic receptors from the early endosome to the plasma membrane in cardiac myocytes. Transient transfection of Rab4 in the HL-1 cardiac muscle cell line remarkably increased the recycling of internalized β2ARs to the plasma membrane with an enhanced signaling as measured by an increased cAMP (cyclic adenosine monophosphate) production. The relationship between augmented expression level of Rab4 and

cardiac hypertrophy has also been found in the Akt2−/− mouse model, which can develop a syndrome similar to the type 2 diabetic cardiomyopathy (Etzion et al., 2010). The up-regulation of Rab4 is found in cardiomyocytes and skeletal muscle but not in the liver or the brain.

1.4 Ubiquitin-Proteasome System

1.4.1 Ubiquitination

Ubiquitin is a highly conserved small protein, comprised of 76 amino acids with a molecular mass of 8.5 kDa. One of the key characteristics of ubiquitin is its C terminal tail with seven lysine residues, where additional ubiquitin molecules attach (Hershko & Ciechanover, 1992).

Ubiquitination, also known as the "kiss of death" process for a protein, is the covalent attachment of ubiquitin to its substrate protein. The ubiquitin-tag signals the substrate protein for internalization and/or degradation in the proteasome (Ciechanover, 1994; Hershko &

Ciechanover, 1998). Enzymes involved in ubiquitination include ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3 (Ciechanover, 2010). Different forms of ubiquitination have been identified (Hochstrasser, 1996; Voges et al., 1999; Hicke, 2001;

Millard & Wood, 2006). Monoubiquitination is characterized as a single ubiquitin attachment to the target protein; multi-ubiquitination refers to when multiple lysine residues within a substrate are modified by a single ubiquitin molecule, and poly-ubiquitination is termed as polymerized ubiquitin chain attachment to the target proteins.

The post-translational modification of proteins by ubiquitin is a critical regulatory mechanism in eukaryotic cells (Hershko & Ciechanover, 1998; Ciechanover, 2010), including cell-cycle progression (Koepp et al., 1999), signal transduction, transcriptional regulation, cell membrane protein endocytosis (Strous et al., 1996; Abriel & Staub, 2005), cellular excitability, cell growth and apoptosis (Robzyk et al., 2000), inflammatory response (Ghosh et al., 1998), and antigen

presentation (Rock & Goldberg, 1999). Two major steps are required in order for protein ubiquitination to occur: (1) signaling and ubiquitin attachment to the protein; and (2) degradation of protein mediated by enzymes. The signaling process of ubiquitin attachment to the substrate is mediated by a series of enzymes, including E1, E2, and E3. In order to be recognized by ubiquitin ligase E3, the substrate must possess a particular sequence (Laney & Hochstrasser, 1999). The tagging of a single ubiquitin molecule or polymerized ubiquitin chain to substrates serves as a signal for the retrograde trafficking (internalization/degradation) of substrates. After the ubiquitin tagging, substrates are targeted for either degradation in proteasomes or other trafficking pathways.

The destination and ubiquitination form of proteins varies. K48 and K63 are the most common lysine residues to form the ubiquitin chain (Millard & Wood, 2006). The long polymerized ubiquitin chain is formed through the K48 residue of ubiquitin (Hochstrasser, 1996; Hershko &

Ciechanover, 1998; Yang & Kumar, 2009), while the short polymerized ubiquitin chain forms through the K63 residue (Hicke, 1999). The former usually targets protein degradation in the 26S proteasome, while the latter mediates DNA repair, protein endocytosis and degradation in the lysosome (Pickart & Eddins, 2004). Specific to the function of monoubiquitination in membrane protein transport, several studies showed that, in mammalian cells, various membrane proteins like ion channels and signal-transducing factors are modulated through monoubiquitination and undergo internalization/degradation (Ciechanover, 1994; Strous & Govers, 1999; Chung et al.,

2001; Sun et al., 2011).

Ubiquitination appears to be a critical process for maintaining the homeostasis of cells.

Abnormality within the ubiquitin system is apparently the cause of many diseases, such as cancer

(Li et al., 2004), diabetes (Sun et al., 1999), inflammation (Layfield & Shaw, 2007),

neurodegenerative disorders (Davies et al., 2007; Upadhya & Hegde, 2007), respiratory diseases

(Sato et al., 2007; Boase et al., 2011), and kidney diseases (Bhalla & Hallows, 2008; Rotin,

2008). For instance, Liddle syndrome is an autosomal dominant disorder characterized by early onset and frequently occurring severe hypertension. The genetically mutated epithelial sodium channel (ENaC) cannot be appropriately degraded due to the loss of the recognition site by the ubiquitin ligase Nedd4-2. Therefore, the increased stability of ENaC on the plasma membrane induces excessive fluid retention in the body, leading to high blood pressure in affected patients

(Knight et al., 2006; Zhou et al., 2007).

1.4.2 Cascade of Ubiquitination

Ubiquitination is carried out by a series of enzymes that activate ubiquitin and transfer it to the target protein for degradation. These enzymes include ubiquitin-activating enzyme E1, ubiquitin- conjugating enzyme E2 and ubiquitin-protein ligase enzyme E3 (Ciechanover, 1994; Hershko &

Ciechanover, 1998; Flores et al., 2003; Abriel & Staub, 2005) (Figure 5). A hierarchy is formed among these enzymes: one E1 serves several E2s, and each E2 serves a large amount of E3s. The prevalence of E1s and E2s is very limited, compared to very significant number of E3s. E3 can recognize a group of substrates with similar ubiquitination signals (Pickart, 2001). To begin the ubiquitination process, the E1 enzyme initially activates ubiquitin at the C-terminal Gly76 in an

ATP-dependent manner to form a thiol ester bond with ubiquitin; inducing transient carrying of active ubiquitin as a thiol ester intermediate by ubiquitin-conjugating enzyme E2, which then interacts with ubiquitin ligase E3 for ubiquitin transfer. Ubiquitin ligase E3s can specifically recognize target proteins and catalyze the formation of an isopeptide bond between target protein lysine residues and the C terminus of the active ubiquitin, inducing the internalization/degradation of the protein (Hershko & Ciechanover, 1992; Pickart, 2001). 21

Ub Ub Ub Ub Ub

Ub E3-HECT Ub E3-RING Ub Ub

E2 Ub

Ub E1 Ub E2 proteasome E1 E1 E3 ATP

Active Ub AMP+PP Protein fragment

Figure 5: Ubiquitination. Ubiquitination is carried out by a sequential action of ubiquitin- activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Free ubiquitins are initially activated by E1s in an ATP-dependent manner. Activated ubiquitins are subsequently transferred to E2s forming an Ub-E2 complex. The Ub-E2 complex can either form a complex with RING domain E3 to ubiquitinate ion channels, or transfer activated ubiquitin to HECT domain E3, which recognize the substrates and directly ubiquitinate ion channels. The ubiquitinated ion channels are targeted for degradation in the proteasome.

1.4.3 Ubiquitin Ligase E3

As described in the ubiquitination cascade, the primary function of ubiquitin ligase E3s is to recognize substrates sharing a particular sequence and catalyze the ubiquitination of target proteins (Pickart, 2001). To date, nine members of the E3 family have been identified in humans, including Nedd4, Nedd4-2 (Nedd4L), ITCH, SMuRF1, SMuRF2, WWP1, WWP2, NedL1, and

NedL2 (Harvey et al., 1999; Pickart, 2001; Passmore & Barford, 2004). They are classified into

HECT domain E3s and RING domain E3s. The ubiquitin ligase Nedd4 (Neural-precursor-cell- expressed developmentally down-regulated 4) belongs to the HECT domain-containing E3 subfamily and has been shown to primarily participate in recognizing membrane proteins for degradation (Pickart, 2001). There are two members of the Nedd4 subfamily in mammalian cells: Nedd4-1 and Nedd4-2. The Nedd4-1 gene (Nedd4) is located on chromosome 15, while the

Nedd4-2 gene (Nedd4L) is located on chromosome 18 (Chen et al., 2001). Nedd4-1 and Nedd4-2 share similar structures and bind to the PPxY or LPSY motifs of its substrates via the WW domains (a 38-40 amino acid module containing two highly conserved tryptophans that bind to proline-rich or proline-containing motifs) (Bedford et al., 2000), with the highest affinity exhibited by WW3 (Harvey et al., 1999; Kanelis et al., 2006; Bruce et al., 2008).

1.4.3.1 Nedd4-1

Nedd4-1 is composed of a C2 domain at the N terminus, three (in mice and rats) or four (in humans) WW domains (protein-protein interaction domain) that bind to the PY motif of its target protein, and an ubiquitin ligase HECT domain at the C terminus that weakly binds to the WW domain within itself (Staub & Rotin, 2006). Nedd4-1 and Nedd4-2 share similar conserved sequences and have overlapping functions. The variable regions located between the WW1 and

WW3 domains can potentially be used to distinguish their functions (Wang et al., 2010). 23

The substrates of Nedd4-1 include ion channels, membrane transporters and membrane receptors, tumor suppressor proteins and proteins experiencing endocytosis (Persaud et al., 2009).

For example, the epithelial sodium channel (ENaC) (Staub et al., 1996b), adaptor proteins

(Morrione et al., 1999), T cells (Heissmeyer et al., 2004), and neuromuscular junctions (Liu et al., 2009) are identified substrates of Nedd4-1. Although Nedd4-1 and Nedd4-2 share target proteins and appear to have overlapping functions, the preferential targets of Nedd4-1 are tumor suppressor proteins and endocytic protein (Katz et al., 2002; Woelk et al., 2006). Although a large amount of research has been done in vitro, concerns regarding the application of Nedd4-1 in vivo were raised due to the fact that in vivo experiments are difficult to control and regulate, raising difficulty in demonstrating the regulatory mechanisms/effects under physiological conditions.

1.4.3.2 Nedd4-2

As mentioned above, Nedd4-2 is also composed of a C2 domain, four WW domains that bind to the PY motif of substrates, and a HECT domain. The structure and related function of Nedd4-2 has also been extensively studied. Nedd4-2 plays a major role in the ubiquitin-proteasome pathway for protein degradation. The preferential target proteins of Nedd4-2 are usually ion channels and membrane transporters (Kimura et al., 2011; Guo et al., 2012; Rotin & Staub,

2012). Different domains within Nedd4-2 have distinct functions in regulating substrate ubiquitination. The initial description of the HECT domain and its function as an ubiquitin ligase was described in studies on E6AP (human papilloma virus E6 Associated Protein) (Scheffner et al., 1993; Huibregtse et al., 1995). Nedd4-2 contains ~350 amino-acids, which mainly participate in accepting activated ubiquitin from E2 and transferring it to substrates (Yang & Kumar, 2009); the C2 domain has an inhibitory effect on the auto-activation of Nedd4-2. The binding of calcium

to the C2 domain can disrupt it from binding to the HECT domain, subsequently activating

Nedd4-2 (Wang et al., 2010). The function of the WW domains is primarily to recognize and bind the ubiquitination signals (a particular motif) in the target substrates.

Nedd4-2 targets several of ion channels, including epithelial sodium channels (ENaC), cardiac voltage-gated sodium channels Nav1.5, hERG channels, KCNQ1 channels, and neuronal voltage- gated sodium channels (Nav) (Fotia et al., 2004; van Bemmelen et al., 2004; Jespersen et al.,

2007; Zhou et al., 2007; Albesa et al., 2011). The effect of Nedd4-2 in the regulation of ENaC has been extensively investigated (Debonneville et al., 2001; Malik et al., 2005; Rotin, 2008).

Nedd4-2 and ENaC interact with each other via direct binding between the WW domain and the

PY motif, which decreases cell surface expression of ENaC through Nedd4-2-mediated monoubiquitination of α, β, and γ subunits of ENaC (Staub et al., 1996a; Fotia et al., 2003; Zhou et al., 2007). The plasma membrane pool but not the intracellular pool of ENaC is selectively degraded, as E2 and E3 interfere only with the internalization/translocation step of cell surface proteins and do not affect newly synthesized proteins exported from the Golgi apparatus

(Debonneville & Staub, 2004). Abnormally regulated ENaC degradation leads to Liddle syndrome, an early–onset hereditary autonomic kidney disease presenting with frequently severe hypertension. It is known that mutations of ENaC in Liddle syndrome disrupt the recognition sites of Nedd4-2, leading to the accumulation of ENaC on the cell membrane and increasing Na+ absorption and consequently water absorption in the distal nephron. Recently, observations in our lab demonstrated that the hERG is also down-regulated by Nedd4-2 (Guo et al., 2012). Like

ENaC, the hERG potassium channel also possesses a PY motif in the C-terminus. Guo et al.

(2012) demonstrated that hERG potassium channels are regulated by the ubiquitin ligase Nedd4-

2. Furthermore, caveolin-3, an integral membrane protein, is involved in the Nedd4-2-mediated

down-regulation of the hERG channel by recruiting Nedd4-2 to the cell membrane, which in turn induces the ubiquitination of hERG channels on the plasma membrane.

One important aspect of the Nedd4-2-subtrate interaction is that Nedd4-2-mediated substrate ubiquitination also results in Nedd4-2 self-ubiquitination and degradation due to the exposure of the PY motif within the HECT domain of Nedd4-2 (Bruce et al., 2008) (Figure 6). Under normal conditions, Nedd4-2 is self-protected by the weak binding between its own WW domains and the

HECT domain. However, when Nedd4-2 encounters its real substrate this weak binding is disrupted, allowing the WW domain to bind to the PY motif of the substrate. The PY motif within the HECT domain of Nedd4-2 is then exposed to the WW domains of neighboring Nedd4-2 molecules, leading to Nedd4-2 self-ubiquitination. The fate of the ubiquitinated Nedd4-2 is unknown. It may follow the endocytic pathway for degradation or be recycled back to further regulate its substrates. To date, most reported studies have focused on the effects of Nedd4-2 on substrate proteins. The regulating mechanism of Nedd4-2 expression levels has not yet been identified.

1.4.3.3 Regulation of Nedd4-1 and Nedd4-2

The activity of Nedd4-1 and Nedd4-2 can be up/down-regulated by many other factors.

Ndfip1(Nedd4 family-interacting protein 1) and Ndfip2 (Nedd4 family-interacting protein 2) act as Nedd4 adaptors in controlling the specificity of target substrates (Shearwin-Whyatt et al.,

2006). In addition, ISG15, an ubiquitin-like protein, can down-regulate the function of Nedd4 by interfering with the interaction between Nedd4 and E2, thereby affecting the catalytic activity of

Nedd4 (Malakhova & Zhang, 2008).

The catalytic activity of Nedd4-2 can also be modified. SGK1, a serine/threonine kinase, can significantly down-regulate the activity of Nedd4-2 by inducing phosphorylation of Nedd4-2 on 26

HECT WW HECT WW HECT

WW PY

Figure 6: Scheme Depicting the Regulation of Nedd4-2 Self-ubiquitination. The WW domains of Nedd4-2 weakly bind to its own HECT domain under normal conditions. After targeting the PY motif of the substrate, Nedd4-2 undergoes a conformational change and the interaction between the HECT domain and the WW domain is disrupted, leading to the exposure of the PY (LPxY) motif within the HECT domain. Exposure of the PY motif causes ubiquitination of Nedd4-2 itself and leads to degradation.

Ser444, and to a lesser extent on Ser338. The phosphorylation of Nedd4-2 can further recruit 14-

3-3 protein, which blocks substrate recognition and targeting (Yang & Kumar, 2009; Chandran et al., 2011). Similar to SGK1, Akt/PKB (protein kinase B) acts on the same Ser residue to induce

Nedd4-2 phosphorylation (Dieter et al., 2004).

1.5 Hypothesis

It has been showen that caveolin-3 and Nedd4-2 are both involved in the down-regulation of hERG expression on the plasma membrane (Guo et al., 2012). However, it is unknown what happens after hERG undergoes endocytosis. Whether internalized hERG channels can be recycled back to the plasma membrane or are directly targeted for degradation is unclear.

Rab4 GTPase is involved in protein recycling from early endosomes to the plasma membrane.

It was also observed that the endogenous expression of Rab4 is altered under some pathological conditions. The expression of endogenous Rab4 is increased in the Akt-/- mouse model, causing developing cardiomyopathy and transgenic overexpression of the β-adrenergic receptor, which leads to heart failure. In addition, Rab4 also mediates the recycling of internalized β-adrenergic receptors; overexpression of Rab4 in mouse myocardium leads to concentric cardiac hypertrophy.

Since ventricular arrhythmias are highly associated with heart failure as well as cardiomyopathy

(Marbán, 1999b; Tomaselli & Marbán, 1999), it is worth investigating the effect of Rab4 on the expression and function of the hERG channel.

Based on current knowledge of the hERG protein regulatory pathway (synthesis vs. degradation), the hypothesis of my project is that overexpression of Rab4 can affect the expression and function of hERG channels. To test this hypothesis, several techniques are used:

HEK cells stably expressing hERG channels (hERG-HEK) and regular HEK 293 cells are used in experiments. Cardiomyocytes isolated from 1-day-old neonatal Sprague-Dawley rats are used to perform experiments in primary cells. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence microscopy and whole-cell patch clamp will be used.

1.6 Objective

The objectives of my project are listed as below:

1. What is the effect of Rab4 on the expression and function of hERG channels in the

hERG-HEK stable cell line?

2. What is the mechanism behind the observed phenomenon?

3. Is the effect of Rab4 applicable in primary cells, such as cardiomyocytes?

Chapter 2 Materials and Methods

2.1 Molecular Biology and Cell Transfection

Human embryonic kidney (HEK) 293 cell line stably expressing hERG channels (hERG-

HEK) was obtained from Dr. Craig January (University of Wisconsin-Madison, WI). The hERG cDNA was obtained from Dr. Gail A. Robertson (University of Wisconsin-Madison, WI). To disrupt the Nedd4-2 interaction with hERG, the ∆1073 (C-terminal truncation mutation) and

Y1078A (point mutation) mutations were generated by Guo et al. using PfuUltra Hotstart PCR

Master Mix (Agilent Technologies, Santa Clara, CA). The mutations were confirmed by DNA sequencing (Eurofins MWG Operon, Huntsville, AL). The human ether-à-go-go (EAG) cDNA was provided by Dr. Luis Pardo (Max-Planck Institute of Experimental Medicine, Göttingen,

+ Germany); Kv1.5 cDNA (encoding the cardiac ultra-rapidly activating delayed rectifier K channel) was provided by Dr. Michael Tamkun (Colorado State University, Fort Collins,

Colorado). All stable cell lines were cultured at 37 ºC in minimum essential medium (MEM, Life

Technology) supplemented with 10% fetal bovine serum (FBS) and 0.4mg/ml G418 (Invitrogen).

The human Nedd4-2 plasmid in pBluescript II was obtained from Kazusa DNA Research Insiute

(Chiba, Japan). The open reading frame of Nedd4-2 was amplified using polymerase chain reaction (PCR) and cloned into HA-pcDNA3 (Invitrogen) to generate HA-tagged Nedd4-2. The plasmid of the catalytically inactive form of Nedd4-2, Nedd4-2-C801S mutant (Nedd4-2CS), was provided by Dr. Hugues Abriel (University of Bern, Switzerland). Human GFP-tagged Rab1,

Rab4, inactive Rab4 mutant Rab4N121I, Rab5, Rab7 and Rab11 plasmids were obtained from

Addgene as well as Dr. Terry Hébert (McGill University). The scrambled control siRNA and

Rab4a siRNA targeting human Rab4a were purchased from Santa Cruz Biotechnology (Santa

Cruz, CA). The Rab4a siRNA targeting rat Rab4a was purchased from Sigma.

For transient transfection, hERG-HEK or HEK 293 cells were grown to a confluence of 60-

70% in a 35-mm dish, and 2 μg of the plasmid of interest was transfected into cells using

Lipofectamine 2000 (Invitrogen). A green fluorescent protein (GFP) plasmid (0.5 μg, pIRES2-

EGFP, Clontech) was co-expressed to identify transfected cells in electrophysiological studies.

After transfection, cells were cultured in 10% FBS-supplemented MEM for 24 h before experiments.

2.2 Neonatal Rat Ventricular Myocyte Isolation

Experimental protocols used for animal studies were approved by the Animal Care Committee of Queen’s University. Cardiomyocytes were isolated from 1-day-old Sprague-Dawley rats of either sex using enzymatic dissociation methods. In brief, approximately 40 hearts were harvested from neonates and cut into small heart bits. The heart bits were transferred to the sterile flask for digestion. For each digestion, 8.5ml PBS solution (supplemented with 10g/L glucose), 3 mg collagenase (240 U/mg), 2.6 mg trypsin (207 U/mg) and 0.63 mg DNase (2987 U/mg) were added. The mixture was gently agitated at 37oC for 10mins. At the end of each digestion, the supernatants containing cells were transferred into a sterile bottle with 20ml Dulbecco’s modified

Eagle’s medium/Ham’s F-12 (DF) medium (Invitrogen, Burlington, OH) supplemented with 20%

FBS. This step was repeated 6 times. Cells were collected and purified using Percoll (GE healthcare). The purified cardiomyocytes were incubated in DF medium supplemented with 10%

FBS for experiments.

Cardiomyocytes were grown on glass coverslips for electrophysiological and immunocytochemical studies, and in 60 mm dishes for Western blot analysis.

2.3 Western Blot Analysis

For Western blot studies, cells were cultured in 35mm dishes at 37 0C for 24h after treatment.

Cells were collected with ice cold phosphate-buffered saline (PBS) and lysed with ice cold RIPA

(Radioimmunoprecipitation) lysis buffer containing the protease inhibitors cocktail (Sigma) and

PMSF (Sigma). The whole-cell lysates were centrifuged at 9000 g for 10 min to pellet insoluble substances. After protein assay to measure supernatant protein concentration, loading samples were made 30 µg/100 µl. Samples of 15µg/lane were separated using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels. Proteins within the gels were transferred onto polyvinylidene difluoride (PVDF) membranes overnight. After transferring, membranes were blocked with 5% skim milk dissolved in Tris-buffered saline (TBS) supplemented with 0.1%

TWEEN 20 for 1 hour and immunoblotted with appropriate primary antibodies. Horseradish peroxidase-conjugated secondary antibodies were used to detect the signals. Proteins were visualized with Fuji film using Enhanced Chemiluminesence (GE healthcare).

Image J and Photoshop were used for quantification. The intensities of proteins in each gel are normalized to their corresponding actin intensities first; the normalized intensities are subsequently compared with the intensity of control cells and described as relative values to their controls.

2.4 Co-Immunoprecipitation (Co-IP)

For co-immunoprecipitation studies, cells cultured in 100mm dishes were collected and lysed as described in Western blot analysis. Each sample containing 500 µg of protein was topped up to 500 µl/tube with water to have a final concentration of 1 µg/µl, and incubated with 2 µg of appropriate primary antibodies for 4 h on a rotator at 4oC. Upon completing the binding between

protein and its corresponding antibody, the mixture was incubated with 35 µl A/G agarose beads at 4oC for another 4h. After incubation, the immunoprecipitate was collected by centrifuging the samples for 10 min at 9000 g. The pellet was washed four times with 800 µl ice-cold RIPA lysis buffer followed by centrifugation at 9000 g for 3mins each time. For the final wash, the supernatant was discarded, and 2X loading buffer containing 5% β-mercaptoethanol was added to the pellet. Samples were boiled for 5 min. Finally, all samples were centrifuged at 10000 RMPs for 10 min in order to obtain the supernatant. The supernatant was analyzed using Western blot analysis. Proteins were immunoblotted with corresponding primary and secondary antibodies.

2.5 Immunofluorescence Microscopy

The hERG-HEK cells were cultured on cover slips and transfected with GFP, GFP-tagged

Rab4, and GFP-tagged Rab4N121I. Twenty-four hours after transfection, cells were rinsed with ice-cold PBS three times and fixed with freshly prepared 4% ice-cold Paraformaldehyde (Alfa

Aesar, USA, dilated in PBS) for 15 min at room temperature. The fixed cells were permeabilized with 0.1% Triton-X 100 for 10 min, and blocked with 5% bovine serum albumin (BSA) dissolved in PBS for 1 h. The permeabilized cells were immunoblotted with rabbit anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody to detect

Nedd4-2. Cells were treated with Prolong anti-fade mounting medium and kept at room temperature. Cultured neonatal rat cardiomyocytes were transfected with GFP-tagged Rab4. Forty eight hours after transfection, cells were fixed and permeabilized. ERG (rat IKr protein) was labeled with anti-hERG primary antibody (C-20) and Alexa Fluor 594- conjugated secondary antibody. The Nedd4-2 was labeled with anti-Nedd4-2 primary antibody and Alexa Fluor 594- conjugated secondary antibody in separate sets of cells. Images were acquired using a Quorum

Wave FX spinning Disc Confocol System with Hamamatsu Orca high resolution camera at the 34

Queen’s University Cancer Research Institute Image Centre. The green fluorescence was generated using an excitation/emission filter of 491 nm/520 nm (+/-20 nm), and the red fluorescence was generated using an excitation/emission filter of 560 nm /590 nm (+/-20 nm).

2.6 Electrophysiological Recording

For electrophysiological studies, whole-cell patch clamp was used for the recording of hERG current (IhERG), EAG current (IEAG), Kv1.5 current (IKv1.5), and IKr. Cells were harvested from dishes by trypsinization and kept in standard MEM at room temperature. Cells were studied within 4 h after harvest. For the recording of the current from the stable cell lines, the standard

+ 135-mM K pipette solution contained (in mM): 135 KCl, 5 EGTA, 1 MgCl2, and 10 HEPES (pH

+ 7.2). The standard 5-mM K bath solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1

MgCl2, 10 glucose, and 10 HEPES (pH 7.4). The sampling frequency is 1000Hz for IhERG recording and 5000Hz for IKr recording. The pipette resistance is between 2.0-3.5 MΩ. For series resistance, it ranges from 3-10 MΩ. The capacitance of hERG-HEK cell line is between 10-15 pF. Only cells transiently transfected with GFP (pIRES2-EGFP, Clontech) and GFP-tagged Rab4

WT were used.

For IhERG recording, the membrane potential was held at -80 mV. Families of hERG currents were evoked with depolarizing steps from -70 mV to +70 mV in 10 mV increments for 4s (Figure

7A). The hERG tail currents were recorded upon a repolarizing step to -50 mV and were used for the analysis of IhERG amplitude. All patch clamp recordings were performed at room temperature

(22±1 °C).

A 70mV B 70mV

-50mV -50mV

-80mV -70mV -80mV -70mV

2s 0.5s

C 70mV

-80mV -70mV -80mV

0.5s

Figure 7: The Protocol for Whole-cell Patch Clamp Current Recording. A, IhERG recording.

The membrane potential was held at -80 mV. The depolarizing steps were between -70 mV and

+70 mV with an increment of 10mV for 4s. The hERG tail current was evoked with a repolarizing step to -50 mV for 5s; B, protocols for EAG current (IEAG) and Kv1.5 current (IKv1.5) recording. The membrane potential was held at -80mV. The depolarizing step was between -

70mV and +70 mV with an increment of 10 mV. The depolarizing step was held for 1s for both

IEAG recording and IKv1.5 recording; C, protocols for IKr recording. The membrane potential was held at -80mV. The current was evoked by depolarization to voltages between -70 mV and 70 mV in 10 mV increments for 1s. The current amplitude upon repolarization to the -80 mV holding potential after the depolarizing step of 50 mV was used to measure the amplitude of native IKr.

For IEAG recording, the membrane potential was held at -80 mV. The series of EAG currents were evoked with depolarizing steps from -70 mV to +70 mV with an increment of 10 mV. Each depolarizing step lasted for 1s. The same protocol was used for IKv1.5 recording (Figure7B).

For the recording of native IKr in cultured neonatal rat cardiomyocytes, the pipette solution contained (in mM): 135 CsCl, 5 MgATP, 10 EGTA, and 10 HEPES with pH 7.2 by CsOH. The bath solution contained (in mM): 135 CsCl, 1 MgCl2, 10 glucose, and 10 HEPES with pH 7.4 by

CsOH. The current was evoked by depolarization to voltages between -70 mV and 70 mV in 10 mV increments (Figure 7C). The current amplitude upon repolarization to the -80 mV holding potential after the depolarizing step of 50 mV was used to measure the amplitude of native IKr

(Zhang, 2006). All patch-clamp experiments were performed at room temperature (22 ± 1°C).

2.7 Reagents and Antibodies

MEM and FBS were purchased from Invitrogen. Rabbit anti-Kv11.1 (hERG), anti-Kv10.1

(EAG-1), anti-ubiquitin, mouse anti-actin antibodies, G418, electrolytes, EGTA, HEPES, glucose, protein synthesis inhibitor cycloheximide (CHX), BSA, and proteasome inhibitor ALLN

(N-acetyl-leu-leu-norleucinal) were purchased from Sigma. Proteasome inhibitor MG132 (N-

CBZ-leu-leu-leucinal) was purchased from EMD Millipore. Goat anti-hERG (N-20 and C-20), rabbit anti-GAPDH, anti-Kv1.5, anti-Rab4 antibodies, Protein A/G PLUS Agarose, goat anti- mouse IgG and goat anti-rabbit IgG, and mouse anti-goat IgG were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Rabbit anti-Nedd4-2 antibody was purchased from Cell

Signaling Technology. Alexa Fluor 594 donkey anti-rabbit secondary antibody was purchased from Invitrogen.

2.8 Statistical Analysis

All data are expressed as the mean ± S.E. One-way analysis of variance followed by

Newman– Keuls post hoc tests (GraphPad Prism), two tailed paired or unpaired Student's t test are used to determine the statistical significance between control and experimental groups. A P value of 0.05 or less was considered significant.

Chapter 3 Results

It is reported that mature hERG channels experience ubiquitination which is mediated by the ubiquitin ligase Nedd4-2, and are targeted for degradation in lysosome (Sun et al., 2011; Guo et al., 2012). However, whether the internalized hERG proteins can be recycled back to the membrane is poorly understood. Since Rab4 GTPase is a molecule located at the early endosome regulating rapid protein recycling, the purpose of my project is to investigate the effect of Rab4 on the expression and function of mature hERG channels on the plasma membrane.

3.1 Overexpression of Rab4 Decreases the hERG Expression Level at the Plasma membrane

hERG protein extracted from the hERG-HEK stable cell line displayed the mature, fully- glycosylated form on the plasma membrane with a molecular weight of 155 kDa, and the immature, core-glycosylated form in the ER with a molecular weight of 135 kDa. Various Rab

GTPases were examined (Rab1, Rab4, Rab5, Rab7 and Rab11) through transient transfection in hERG-HEK cells for 24h. Figure 8 shows that Rab4 significantly reduced the expression level of mature 155-kDa hERG without affecting immature 135-kDa hERG (n=3-5). This finding is interesting because if Rab4 is involved in rapid recycling of hERG after internalization, an increase of the 155-kDa hERG expression level is expected.

3.2 Overexpression of Rab4 Decreases the hERG Current (IhERG)

Consistent with the Western blotting result, overexpression of Rab4, but not inactive

Rab4N121I decreased IhERG by 40% compared with control (Ctrl) (Figure 9). While Rab4 decreased the IhERG amplitude, it did not affect the biophysical properties of the hERG channel.

155 kDa hERG 135 kDa

Actin 42 kDa 1.5 1.0

0.5 ** (155 kDa) (155

Intensity-Rel 0.0

Figure 8: Rab4 Decreases the Expression Level of Mature hERG Protein on the Plasma membrane. Effects of various Rab GTPases on hERG expression levels. hERG-HEK cells were transiently transfected with various Rab GTPases for 24h. Whole-cell protein was obtained and analyzed using Western blot. The relative band intensities (Intensity-Rel) of the 155-kDa hERG bands from cells transiently transfected with various Rab GTPases were normalized to the value of the control (Ctrl) cells in each gel, summarized and plotted against each treatment (n=3-5). **

P<0.01 vs. control.

Ctrl Rab4 Rab4N121I

nA 1

2 s Ctrl 1.5 Rab4 Rab4N121I

1.0

0.5 **

Tail current (nA) current Tail 0.0 -60 -30 0 30 60 Voltage (mV)

Figure 9: hERG current (IhERG) is Decreased by Rab4 Overexpression. Representative hERG currents recorded from pcDNA3- (Ctrl), Rab4-or inactive Rab4N121I-transfected cells. hERG-

HEK cells grown on the coverslip were transiently transfected with pcDNA3+GFP, GFP-tagged

Rab4 and GFP-tagged inactive Rab4N121I, respectively, for 24h. Only cells expressing green fluorescence were selected for recording. Families of hERG current were recorded upon depolarizing steps between -70mV and 70mV with an increment of 10mV. Tail currents were elicited by a repolarizing step to -50mV. The tail current recording at a depolarizing step of

50mV was used to analyze IhERG amplitude. The summarized tail current-voltage relationships are shown beneath the current traces. (n=8 for Ctrl and Rab4, n=4 for Rab4N121I). The data were collected from at least three independent experiments. ** P<0.01 vs. control.

The half activation voltage and the slope factor of IhERG were −3.1±0.3 mV and 7.1±0.3 mV in control cells, and −6.5±0.9 mV and 7.9±0.7 mV in Rab4- transfected cells.

3.3 The effect of Rab4 is Specific to hERG Potassium Channels

Overexpression of Rab4 neither affected the expression level of Kv1.5 nor EAG channels stably expressed in HEK 293 cells (n=3, respectively) (Figure 10 A). Consistent with the data obtained from Western blot analysis, Rab4 did not affect IKv1.5 and IEAG (Figure 10 B). Thus, among potassium channels Kv1.5, EAG and hERG, Rab4 selectively targets the hERG channel.

3.4 Knockdown of Rab4 Expression Increases hERG Protein Expression

The HEK 293 stable cell line endogenously expresses Rab4. To further confirm the effect of

Rab4 on the expression of hERG channels, we used Rab4 siRNA to knockdown the expression of endogenous Rab4. As shown in Figure 11, knockdown of Rab4 significantly increased the expression of hERG protein in the hERG-HEK cell line.

3.5 Rab4 Increases hERG Ubiquitination

Ubiquitination is a process where proteins are tagged with ubiquitin for internalization and degradation in lysosomes/proteasomes (Ciechanover, 1994; Strous & Govers, 1999). We and another groups both suggested that the ubiquitination and degradation of hERG is mediated by the ubiquitin ligase Nedd4-2 (Albesa et al., 2011; Guo et al., 2012). To analyze whether the

Rab4-induced mature hERG reduction affects hERG ubiquitination, I performed a co-IP experiment. Whole-cell proteins were extracted from hERG-HEK cells 24 h after pcDNA3

(control) or Rab4 transfection with the presence of the proteasome inhibitor, ALLN (50 μM), to impede protein degradation. Ub and its associated proteins were immunoprecipitated with an anti-

A B EAG Kv1.5

75 Ctrl EAG 130 Kv1.5

68 5nA

0.5nA 0.5 s 0.5 s Rab4-GFP 50 Rab4-GFP 50

Actin 42 Actin 42 Rab4

68 kDa

75 kDa

1.0 1.0 (10) (12) (21) (30) )

) 2 10

0.5 0.5 nA

( (

1 5 (130kDa)

1 2

Intensity-Rel 0.0 0.0 EAG

Intensity-Rel

I Kv1.5

0 I 0

Figure 10: Overexpression of Rab4 Neither Affects the Expression nor the Function of

Kv1.5 and EAG channels. A, representative Western blot images of the expression of Kv1.5 and

EAG channels in the presence or absence of Rab4. The band intensities of respective channel proteins from Rab4-transfected cells were normalized to the values from control cells and summarized (n=3, respectively). B, effects of Rab4 on the EAG and Kv1.5 currents. EAG and

Kv1.5 currents with or without the presence of Rab4 are shown along with the current amplitudes upon the 50mV depolarization step. For both Western blot analysis and whole cell recording,

HEK cells stably expressing EAG channels or Kv1.5 channels were transiently transfected with pcDNA3+GFP or GFP-tagged Rab4, respectively, for 24h. Cells expressing green fluorescence were selected for current recording. The numbers in the parentheses above each bar represent number of cells tested from at least three independent experiments.

155 hERG 135

Actin 42

Rab4 25

1.5 ** 1.0

0.5

(155 kDa) 0.0 Intensity-Rel

Ctrl siRNA Rab4 siRNA

Figure 11: Knockdown of Endogenous Rab4 Increases the Expression of hERG Channels in hERG-HEK Cells. hERG-HEK cells were transiently transfected with control siRNA or human

Rab4 siRNA for 24h. Whole-cell lysate was obtained and analyzed using Western blot analysis

(n=4). The 155-kDa band intensities are shown as relative values to its control. ** P<0.01 vs. control. The knockdown rate of Rab4 is 70% (data not shown).

Ub antibody and hERG expression was detected. As shown in Figure 12, the ubiquitination of hERG was significantly increased by Rab4 overexpression, as a more intense ubiquitinated hERG band higher than the 155-kDa band in Western blot control was detected in Rab4 overexpressed cells. This result suggested that Rab4 overexpression increased ubiquitination of hERG on the plasma membrane.

3.6 The Interaction Between hERG and Nedd4-2 is Increased by Rab4 Overexpression

Since Nedd4-2 mediates the ubiquitination of hERG, I wanted to investigate whether the interaction between Nedd4-2 and hERG is also increased under Rab4 overexpression. To examine the hERG-Nedd4-2 interaction, I again performed co-IP experiments. Rab4 overexpression also significantly enhanced the hERG-Nedd4-2 interaction as evidenced by a stronger Nedd4-2 band in anti-hERG antibody-precipitated proteins from Rab4-transfected cells compared with that from control cells (Figure 13). These results suggested that the reduction of mature hERG on the plasma membrane induced by Rab4 is associated with increased hERG ubiquitination and interaction with Nedd4-2.

3.7 Disrupting the hERG-Nedd4-2 Interaction Eliminates the Effect of Rab4 on hERG

Channels

To determine if Nedd4-2 plays a role in Rab4-induced reduction of hERG expression on the plasma membrane, we examined the effect of Rab4 on two mutant hERG channels, ∆1073

(Nedd4-2 binding site is truncated) and Y1078A (Nedd4-2 binding site is modified). As shown in

Figure 14 A, disruption of Nedd4-2 binding site on hERG mutants completely abolished the effect of Nedd4-2 on hERG. Interestingly, the effect of Rab4 was also impeded in these two

hERG 155 kDa hERG 155 kDa

50 kDa IgG IgG 50 kDa

IP: Ub GAPDH IP: Ub IB: hERG IB: hERG

Figure 12: Rab4 enhances hERG ubiquitination. hERG-HEK cells were transfected with pcDNA3 (Ctrl) or Rab4 for 24 h. The cells were then treated with the proteasome inhibitor ALLN

(50 μM) for 24 h to prevent hERG degradation. The whole-cell lysates were precipitated with an anti-Ub antibody and immunoblotted with an anti-hERG antibody (n=7). To confirm that Rab4 enhanced the Ub-hERG interaction, GAPDH was used as a control for the anti-Ub antibody (left panel) in the immunoprecipitation assay. As well, HEK cells were used as control for hERG-

HEK cells (right panel). The hERG immunoprecipitated by the anti-Ub antibody that is slightly higher than the Western blot control hERG expression represents monoubiquitinated hERG protein.

Nedd4-2 110

IgG 50

IP: hERG IB: Nedd4-2

Figure 13: Rab4 Increases the Interaction between Nedd4-2 and the hERG Channel. The hERG-HEK cells were transfected with pcDNA3 (Ctrl) or Rab4 for 24 h. Whole-cell lysate was immunoprecipated with an anti-hERG antibody (N-20) and immunoblotted using an anti-Nedd4-2 antibody (n=3).

A WT ∆1073 Y1078A B WT ∆1073 Y1078A

155 155 hERG hERG 135 135

Nedd4-2 110 Rab4-GFP 50

Actin 42 Actin 42

1.0 1.01.0

1.0 Rel

Rel

0.50.5 ** 0.50.5 ** *

0.0 0.0 0.0

Intensity

0.0 band) (upper

Intensity (upper band) (upper

WT 1073 Y1078A WT 1073 Y1078A

Figure 14: Nedd4-2 is Involved in the Rab4-mediated Reduction of Mature hERG

Channels. A, effects of Nedd4-2 on the expression of WT and mutant hERG channels. hERG-

HEK cells were transfected with pcDNA3 (Ctrl) or Nedd4-2 for 24h. The expression of hERG and Nedd4-2 were detected. Disrupting the Nedd4-2 binding site in hERG eliminated the Nedd4-

2-induced reduction in the mature (upper) hERG expression (n=4). B, effects of Rab4 on the expression of WT and mutant hERG channels. hERG-HEK cells were transfected with pcDNA3

(Ctrl) or GFP-tagged Rab4 for 24h. The expression of hERG and GFP were detected. Disrupting the Nedd4-2 binding site in hERG completely abolished the Rab4-induced reduction of the mature hERG expression (n=7). In both A and B, the relative band intensities of the mature hERG from Nedd4-2 or Rab4-transfected cells was normalized to their respective controls and summarized. ** P<0.01 vs. control.

mutant hERG channels, indicating that the effect of Rab4 on mature hERG is Nedd4-2 dependent

(Figure14 B).

3.8 Overexpression of Rab4 Increases the Endogenous Nedd4-2 Expression Level

The ubiquitin ligase Nedd4-2 is expressed in HEK 293 cells endogenously. To investigate the mechanisms of Nedd4-2 in the Rab4-mediated reduction in hERG expression, we examined the direct effect of Rab4 on the endogenous Nedd4-2. Western blot and immunofluorescence microscopy were performed. As the Western blot analysis showed, overexpression of Rab4 significantly enhanced the Nedd4-2 expression level, whereas the inactive Rab4N121I had no effect (Figure 15A). The overexpressed GFP-tagged Rab4 presented a molecular weight of 50 kDa. To further confirm the effects of Rab4 on Nedd4-2 expression, we also knocked down Rab4 using siRNA transfection. Knockdown of Rab4 in HEK cells resulted in a significant decrease in the Nedd4-2 expression level (Figure 15B). For immunofluorescence microscopy experiments, hERG-HEK cells were transfected with GFP, GFP-tagged Rab4, and GFP-tagged Rab4N121I for

24h to show green fluorescence. Nedd4-2 detection with Alexa Fluor 594 donkey anti-rabbit secondary antibody shows red fluorescence. Consistent with the Western blot analysis, cells transfected with GFP-tagged Rab4 showed an increased level of Nedd4-2 expression compared with untransfected cells. The GFP-tagged Rab4N121I did not affect the level of Nedd4-2 (Figure

16).

3.9 Rab4 Increases Nedd4-2 Expression by Affecting the Nedd4-2 Degradation Rate

The increased expression level of Nedd4-2 could be induced by two possibilities: increased biosynthesis or decreased degradation rate. Bruce et al. (2008) suggested that the PY motif located within the HECT domain of Nedd4-2 is important for maintaining the stability of Nedd4-

A 1.5 **

Nedd4-2 110 kDa 1.0 0.5

Rab4-GFP 50 kDa kDa) (110 Intensity-Rel 0.0 Actin 42 kDa Ctrl Rab4

Rab4 N121I B 1.0 *

Nedd4-2 110 kDa 0.5 (110 kDa) (110

Actin 42 kDa Intensity-Rel 0.0

Rab4 25 kDa Ctrl siRNA Rab4 siRNA

Figure 15: Effect of Overexpressed Rab4 on Endogenous Nedd4-2 Expression. A: effect of

Rab4 and inactive Rab4N121I on the expression level of Nedd4-2. The hERG-HEK cells were transfected with pcDNA3, GFP-tagged Rab4 or Rab4N121I. Twenty four hours after transfection, whole-cell lysate was extracted and analyzed using Western blot analysis (n=4). Anti-GFP antibody was used to detect Rab4 overexpression. B: knockdown of Rab4 decreases the expression of endogenous Nedd4-2. The hERG-HEK cells were transfected with scrambled control siRNA and Rab4 siRNA. Twenty four hours after transfection, whole-cell lysates were extracted and analyzed using Western blot analysis (n=4). Rab4 was detected using an anti-Rab4 antibody. For analyses, the intensities of the 110-band of Nedd4-2 from cells in experimental groups were normalized to their respective controls and plotted as relative values. * P< 0.05, **

P<0.01 vs. control.

GFP Nedd4-2 Merge DIC

EGFP

GFP

Rab4

GFP -

10 µm Rab4N121I

Figure 16: Confocal Image Showing Rab4 Increases the Endogenous Expression of Nedd4-

2. Immunofluorescence microscopy showing overexpression of Rab4 but not inactive Rab4N121I enhances the expression of endogenous Nedd4-2. hERG-HEK cells were transfected with GFP,

GFP-tagged Rab4 or GFP-tagged Rab4N121I (green). Nedd4-2 was labelled with anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated (red) secondary antibody.

2 itself. When Nedd4-2 is free in the cytoplasm, the PY motif of Nedd4-2 weakly binds to the

WW domain of the same molecule, protecting it from being self-ubiquitinated. However, after targeting its substrates, Nedd4-2 exposes its PY motif to the WW domains of neighboring Nedd4-

2 molecule, leading to self-ubiquitination and degradation (Wang et al., 2010).

To test the possibility that Rab4 increases the expression of Nedd4-2 by interfering with its degradation, cells transfected with pcDNA3 (Ctrl) and Rab4 were treated with the proteasome inhibitor ALLN (50 μM) (or 10 μM MG132, data not shown) to prevent protein from degradation.

We have previously shown that proteasome inhibition can impede Ub-mediated degradation of hERG channels (Sun et al., 2011). As shown in Figure 17, treatment of hERG-HEK cells with 50

μM ALLN (or 10 μM MG132, data not shown) increased the level of Nedd4-2 expression, likely by preventing proteasomal degradation. On this elevated background, Rab4 overexpression no longer significantly increased Nedd4-2 expression levels.

3.10 Rab4 Impedes the Degradation Rate of Nedd4-2

As the previous result showed, Rab4 mimics the effect of inhibiting proteasome activity. To further investigate the mechanisms of the Rab4-impeded degradation rate of Nedd4-2, I monitored the degradation rate of Nedd4-2 in both control (pcDNA3) and Rab4-transfected cells over a period of time. Both groups of cells were treated with cycloheximide (CHX, 10 μg/ml), to inhibit newly synthesized protein, and were collected at the indicated time points. As shown in

Figure 18, the decay rate of Nedd4-2 in the Rab4-overexpressed cells was slower than that from the control group. While the Nedd4-2 expression decreased by more than 50% after 12 h with

CHX treatment in control cells, it only decreased 15% in Rab4-transfected cells.

ALLN

120 kDa Nedd4-2 110 kDa

Rab4-GFP 50 kDa

Actin 42 kDa

* N.S.

2.0 )

Rel

kDa 1.0

(110 (110 Intensity 0.0

ALLN

Figure 17: Inhibition of Nedd4-2 Degradation Eliminates the Rab4-mediated Increase in

Nedd4-2 Expression. HEK cells were transfected with pcDNA3 (Ctrl) or Rab4 for 24h with or without the treatment of the proteasome inhibitor ALLN (50 µM). Cells were then collected and subjected to Western blot analysis. Anti-GFP antibody was used to detect Rab4 expression. The intensities of the 110-kDa Nedd4-2 from cells transfected with Rab4 were normalized to its own control and were plotted (n= 4). * P<0.05 vs. control. N.S.: no significant difference.

CHX (h) 0 3 6 9 12

Nedd4-2 120 110 Ctrl

Actin 42

120 Nedd4-2 110 Rab4 Actin 42

1.0 **

** Rab4

Rel -

0.5 Ctrl

kDa) (110 Intensity 0.0

0 3 6 9 12 CHX Chase Time (h)

Figure 18: Rab4 Interferes with the Degradation Process to Increase Nedd4-2 Expression.

Rab4 slows down the degradation rate of Nedd4-2. Data showing the expression levels of Nedd4-

2 at various time points following cycloheximide (CHX) (10µg/ml) treatment of hERG-HEK cells transfected with pcDNA3 (control) or pcDNA3-encoding Rab4. The relative intensities of the 110-kDa Nedd4-2 bands were normalized to its own control value at time 0 and were plotted versus time (h). (n=4). ** P<0.01 vs. control.

3.11 Catalytic Activity of Nedd4-2 is Necessary for Rab4-mediated Enhancement of Nedd4-

Given that Nedd4-2 is an ubiquitin ligase and demonstrates self-ubiquitination and degradation,

I wanted to test whether the self-ubiquitination and catalytic activity is required for the altered degradation rate by Rab4.

The ubiquitination level of WT Nedd4-2 and catalytically inactive Nedd4-2 mutant, Nedd4-2-

C801S were analyzed using co-immunoprecipitation. Consistent with the observation of Bruce et al. (2008), the WT Nedd4-2, but not the catalytically inactive Nedd4-2, experienced significant ubiquitination as evidenced by the strong band precipitated by Ub in Nedd4-2 overexpressed cells

(Figure 19 A). In particular, besides the smeared background, which may indicate polyubiquitination, a single band slightly higher than the Nedd4-2 band of Western blot analysis is apparent, which should reflect the monoubiquitinated Nedd4-2.

To further investigate the effect of Rab4 on both WT Nedd4-2 and catalytically inactive

Nedd4-2-C801S. HEK cells transfected with GPF-tagged Rab4 were co-transfected with WT

Nedd4-2 and Nedd4-2-C801S. The expression of Nedd4-2 and Nedd4-2C801S were examined.

The catalytic activity appears to be a prerequisite for the Rab4-mediated increase in Nedd4-2 expression, due to the fact that while Rab4 significantly increased the expression level of overexpressed WT Nedd4-2, it failed to increase the expression level of Nedd4-2-C801S (Figure

19 B). These data raised the possibility that Rab4 preferentially interacts with catalytically active

Nedd4-2, interferes with its degradation and promotes its recycling, all together resulting in an increased expression level of Nedd4-2.

3.12 Rab4 Specifically Interacts with Catalytically Active Nedd4-2

If Rab4 only increases the expression of catalytically active Nedd4-2 not inactive Nedd4-2- 55

A B Nedd4-2 Nedd4-2CS

Nedd4-2 110 kDa Nedd4-2 110 kDa Rab4-GFP 50 kDa Actin 42 kDa IgG 50 kDa * Nedd4-2

2.0 Nedd4-2CS

Rel -

1.0Title Axis Y IP: GAPDH Ub

(110 kDa) (110 0.0

IB: Nedd4-2 Intensity X Axis Title

Figure 19: The Rab4-mediated Enhancement of Nedd4-2 is Dependent on the Catalytic

Activity of Nedd4-2. A, ubiquitination analysis of WT Nedd4-2 and catalytically inactive Nedd4-

2C801S. Whole-cell lysates from HEK cells transfected with Nedd4-2 or Nedd4-2C801S were immunoprecipitated with an anti-Ub antibody. The precipitates were immunoblotted with an anti-

Nedd4-2 antibody to detect ubiquitinated Nedd4-2 (n=3). A fraction of protein from Nedd4-2- transfected cells was immunoblotted with an anti-Nedd4-2 antibody to show Nedd4-2 expression as a positive control. B, effects of Rab4 overexpression on the expression of Nedd4-2 and catalytically inactive Nedd4-2C801S. Cells transfected with pcDNA3 (Ctrl) or Rab4 were co- transfected with either Nedd4-2 or Nedd4-2C801S. Western blot analysis was performed 24 h after transfection. The intensities of Nedd4-2 in Rab4 transfected cells were normalized to the control and expressed as relative values (n=3). * P<0.05 vs. Ctrl.

C801S, it is possible that Rab4 preferentially interacts with ubiquitinated active Nedd4-2 to interfere with its degradation and promote its recycling, resulting in an increased expression level.

To examine the interaction, a co-immunoprecipitation experiment was performed. HEK293 cells transfected with Rab4 were co-transfected with Nedd4-2 and Nedd4-2-C801S for 24h. An anti-

Rab4 antibody was used to immunoprecipitate Rab4 and its associated protein from whole-cell protein. The expression of Nedd4-2 was detected. As shown in Figure 20, between Nedd4-2 and

Nedd4-2C801S, Rab4 interacts with Nedd4-2 as revealed by a strong band detected in Nedd4-2 transfected cells. Furthermore, the Nedd4-2 band that interacts with Rab4 is slightly higher than the Nedd4-2 band of the Western blot control, suggesting that Rab4 possibly interacts with monoubiquitinated Nedd4-2.

3.13 Rab4 Decreases the Ubiquitination of Nedd4-2

Rab4 specifically interacts with WT Nedd4-2 and inhibits its degradation rate as shown in

Figure 18 and Figure 20. Given the fact that Rab4 mediates rapid recycling, Rab4 overexpression can potentially decrease the amount of ubiquitinated Nedd4-2 that is targeted for degradation by increasing the recycling rate. A co-IP experiment was performed to analyze the ubiquitination of

Nedd4-2 in the presence or absence of Rab4. HEK cells transfected with Rab4 were co- transfected with Nedd4-2 and Nedd4-2-C801S for 24h. Ubiquitin was immunoprecipitated using an anti-ubiquitin antibody, and its associated protein was detected. As shown in Figure 21, the expression of ubiquitinated Nedd4-2 was significantly reduced by Rab4 overexpression compared with control (pcDNA3), suggesting that Rab4 slows the degradation rate, and enhances the expression of Nedd4-2.

Nedd4-2 110 kDa

IgG 50 kDa

IP: GAPDH Rab4

IB: Nedd4-2

Figure 20: Rab4 Interacts with Catalytically Active Nedd4-2. Rab4 preferentially interacts with Nedd4-2 relative to Nedd4-2C801S. Lysates from HEK cells transfected with Nedd4-2 or

Nedd4-2C801S were immunoprecipitated using an anti-Rab4 antibody. The precipitates were immunoblotted with an anti-Nedd4-2 antibody (n=4). A fraction of protein from Nedd4-2- transfected cells on the left column was also immunoblotted with an anti-Nedd4-2 antibody to show Nedd4-2 expression as a positive control.

Nedd4-2 110 kDa

IgG 50 kDa

IP: GAPDH Ub IB: Nedd4-2

Figure 21: Rab4 Decreases the Ubiquitination of Nedd4-2. HEK 293 cells transfected with pcDNA3 or Rab4 were also co-transfected with Nedd4-2. An anti-Ub antibody was used to immunoprecipitate ubiquitin and its associated proteins from whole-cell lysates. The expression of Nedd4-2 was detected (n=6). A fraction of protein was also immunoblotted with an anti-

Nedd4-2 antibody as a positive control. A fraction of protein from Nedd4-2-transfected cells was immunoblotted with an anti-Nedd4-2 antibody to show Nedd4-2 expression as a positive control.

3.14 Effect of Rab4 on the Expression of the ERG Channel in Neonatal Rat Cardiomyocytes

Rab4 is natively expressed in cardiomyocytes and serves important functions. By transgenic expression of dominant negative Rab4 S27N in mice, Odley et al. (2004) showed that Rab4 mediates recycling of internalized β adrenergic receptors, which are necessary for normal cardiac catecholamine responsiveness and resensitization after agonist exposure. The Rab4 expression level is altered in certain pathological conditions. Transgenic overexpression of β2- adrenergic receptors in mouse hearts leads to heart failure with augmented Rab4 expression levels (Wu et al., 2001). Increased Rab4 expression is also observed in Akt2-deficiency-induced cardiomyopathy which is similar to type 2 diabetic cardiomyopathy (Etzion et al., 2010).

Transgenic overexpression of Rab4 in the mouse myocardium induces concentric cardiac hypertrophy (Filipeanu et al., 2006).

To determine whether Rab4 expression affects ERG channel, I altered Rab4 expression levels by overexpressing Rab4 as well as knocking down Rab4 in neonatal rat cardiomyocytes. As shown in Figure 22A, B and C, overexpression of Rab4 decreased, whereas knockdown of Rab4 increased, the mature form of ERG (IKr protein in rats) in neonatal rat cardiomyocytes.

3.15 Effect of Rab4 on the Function of IKr in Neonatal Rat Cardiomyocytes

To further confirm that the IKr conducted by ERG channels in neonatal rat cardiomyocytes was also regulated by Rab4, GFP-tagged Rab4 was overexpressed in neonatal rat cardiomyocytes for

48h. Cells expressing GFP were selected for electrophysiological studies. As shown in Figure 23, overexpression of Rab4 significantly decreased the IKr recorded by whole-cell patch clamp using

+ + Cs to isolate IKr from other K currents.

A B

155 150 155 ERG150 ERG 130 130 135 135

Rab4-GFP 50 Rab4 25

Actin 42 Actin 42

C 2.0 C * 1.5 * 1.5 1.0 1.0 * * 0.5 0.5 (150 kDa) Intensity-Rel 0.0 0.0 Rab4 pCDNA3 Ctrl siRNA Rab4 siRNA

Figure 22: Effect of Rab4 on the Expression of ERG in Neonatal Rat Cardiomyocytes. A and

B: overexpression of Rab4 decreases whereas knockdown of Rab4 increases the expression of

ERG in neonatal rat cardiomyocytes. Cultured neonatal rat cardiomyocytes were transfected with pcDNA3 or Rab4-GFP; control siRNA or Rab4 siRNA. Forty eight hours after transfection, whole-cell lysate was extracted and analyzed using Western blot analysis. In the right lane of both

A and B, hERG extracted from hERG-HEK cells were loaded as a positive control. C, summarized relative intensities (Intensity-Rel) of the mature ERG band compared to their respective controls (n=4-6, * P<0.05).

D Ctrl Rab4

0.1 nA 0.5 s

0.1 *

Kr-Cs I (nA) I 0.0 Ctrl Rab4

Figure 23: Effect of Rab4 on IKr in Neonatal Rat Cardiomyocytes. Overexpression of Rab4 decreases IKr. Neonatal rat cardiomyocytes were transfected with GFP or GFP-tagged (Rab4-

+ GFP). Cells expressing GFP were selected for recording Cs -mediated IKr (IKr-Cs). The tail current was recorded upon a repolarizing step to -80mV and was used to analyze the current amplitude.

The summarized amplitudes of the tail currents of IKr-Cs were plotted (n=21 for control and 22 for

Rab4-transfected cells). * P<0.05 vs. control.

3.16 Immunofluorescence Microscopy Revealing the Effects of Rab4 on the

Expression of Both the ERG Channel and Nedd4-2 in Neonatal Rat Cardiomyocytes

I have shown that hERG channel in stable cell line are regulated by Rab4 via the activity of

Nedd4-2. To confirm the involvement of Nedd4-2 in Rab4-mediated changes in ERG, immunofluorescence microscopy was used to examine the effects of Rab4 overexpression on

ERG and Nedd4-2 levels in neonatal rat cardiomyocytes. As shown in Figure 24, overexpression of Rab4-GFP led to a decrease in ERG expression and an increase in Nedd4-2 expression.

Figure 24: Effect of Rab4 on the Expression of ERG and Nedd4-2 in Neonatal Rat

Cardiomyocytes. Confocal image showing that overexpression of Rab4 decreases the native

ERG expression (top panel) and increases Nedd4-2 expression (bottom panel). Neonatal rat cardiomyocytes were transfected with GFP-tagged Rab4 (green). The ERG was labeled with an anti-C20 primary antibody and Alexa Fluor 594-conjugated (red) secondary antibody. The

Nedd4-2 was labeled with an anti-Nedd4-2 primary antibody and Alexa Fluor 594-conjugated

(red) secondary antibody. Detection of ERG (Red) or Nedd4-2 (Red) was performed independently.

Chapter 4 Discussion

In the present study, I investigated the effect of Rab4 GTPase on the expression and function of the hERG potassium channel in the hERG-HEK stable cell line and neonatal rat cardiomyocytes. A novel regulatory mechanism was found that Rab4 decreases the expression of hERG channels on the plasma membrane through enhancing the expression level of the ubiquitin ligase Nedd4-2, which has been shown to mediate hERG channel ubiquitination and degradation

(Albesa et al., 2011; Guo et al., 2012).

As mentioned, Rab4 GTPase is a molecule that regulates the rapid recycling of proteins from the early endosome back to the plasma membrane. The initial intention of my project was to determine whether Rab4 can upregulate hERG expression on the plasma membrane. Surprisingly, instead of increasing hERG on the plasma membrane, Rab4 significantly decreases both the expression of hERG on the plasma membrane and IhERG (Figure 8 and Figure 9). However, the gating properties (open vs. closed) are not affected by Rab4 overexpression (Figure 9).

As hERG expression is decreased by Rab4, and Nedd4-2 is one of the molecules that mediate hERG reduction, we investigated the direct effect of Rab4 on the expression of Nedd4-2.

Surprisingly, we found that the expression of Nedd4-2 is increased by Rab4 overexpression

(Figure 15), which further increases the ubiquitination and degradation of hERG protein on the plasma membrane (Figure 12). The direct effect of Rab4 on hERG recycling is negligible, due to the fact that overexpression of Rab4 has no effect on hERG expression in the Nedd4-2 binding site disrupting mutants ∆1073 and Y1078A, indicating that Nedd4-2-mediated regulation is the primary pathway for Rab4 to regulate hERG (Figure 14B).

Ubiquitination is a process targeting proteins for degradation. A covalent bond is formed between single or multiple ubiquitin molecules and a target protein, inducing ubiquitinated proteins for internalization and degradation in the proteasome (Ciechanover, 1994). Bruce et al.

(2008) suggested that the PY motif within the HECT domain of Nedd4-2 is responsible for the stability and inhibition of auto-ubiquitination of Nedd4-2 itself. Without the presence of other substrates, the PY-motif (LPXY) of Nedd4-2 weakly binds to the WW domains of Nedd4-2 itself to prevent Nedd4-2 from being ubiquitinated by adjacent Nedd4-2 molecules. However, when

Nedd4-2 interacts with its target proteins, such as hERG and ENaC, Nedd4-2 undergoes a conformational change, exposing the PY-motif to adjacent Nedd4-2 molecules and leading to the ubiquitination and degradation of Nedd4-2. Our data demonstrated that Nedd4-2 indeed undergoes self-ubiquitination, whereas the catalytically inactive Nedd4-2-C801S had a significantly lower degree of self-ubiquitination (Figure 19A). Rab4 overexpression significantly slows down the degradation rate of Nedd4-2 and increases the expression of Nedd4-2 but not

Nedd4-2C801S (Figure 18 and Figure 19B), indicating that catalytic activity is critical to the

Rab4-mediated increase of Nedd4-2. However, in contrast to the overexpressed Nedd4-2 shown in Figure 14 and Figure 19B, endogenous Nedd4-2 demonstrates two bands with a molecular mass of 110 kDa and 120 kDa as shown in Figure 15, 17, and 18. Two reasons prompted me to focus on the 110-kDa Nedd4-2. Firstly, the molecular weight of the overexpressed Nedd4-2 is

110 kDa, which has already been shown to mediate hERG ubiquitination and degradation (Albesa et al., 2011; Guo et al., 2012). Secondly, co-IP experiments investigating the hERG-Nedd4-2 interaction revealed that only the 110-kDa Nedd4-2 interacts with the mature hERG band (Figure

13). Taken together, these findings support the notion that Rab4 mediates catalytically active

Figure 25: A Proposed Scheme Depicting How Rab4 Increases the Nedd4-2 Expression

Level by Facilitating Nedd4-2 Recycling. Prior to targeting its substrates, Nedd4-2 remains inactive with its WW domain binding to the HECT domain of the same molecule. The binding between the PY motif of hERG channels on the plasma membrane and the WW domain of

Nedd4-2 leads to Nedd4-2 activation, which mediates hERG ubiquitination and degradation.

Meanwhile, activation of Nedd4-2 leads to a conformational change which exposes its PY motif within the HECT domain to other Nedd4-2 molecules, leading to the ubiquitination of Nedd4-2.

The ubiquitinated Nedd4-2 can be either degraded along the degradation pathways or sorted in the early endosomes and recycled back to the plasma membrane. Rab4 facilitates the recycling and consequently reduces degradation of Nedd4-2, resulting in an increased Nedd4-2 level. The increased Nedd4-2 level further causes a decreased expression of mature hERG channels at the plasma membrane.

Nedd4-2 recycling, and that deubiquitination likely also occurs during the recycling process

(Figure 25).

The increased Nedd4-2 further targets hERG on the plasma membrane through the proline- rich PY motif, the targeting site of the WW domain of Nedd4-2, of substrates such as ENaC and hERG. Binding between Nedd4-2 and its substrate results in the transferring of activated ubiquitin from Nedd4-2 to the substrate, and causes ubiquitination of the substrate (Staub et al.,

1996a; Flores et al., 2003). It is worth noticing that although both immature (located at the ER with a molecular weight of 135kDa) and mature (located on the plasma membrane with a molecular weight of 155kDa) hERG channels both possess the PY motif, our data show that

Nedd4-2 selectively targets and induces degradation of the 155-kDa (mature) hERG channels.

Similar results were demonstrated in the Nedd4-2-mediated ubiquitination and degradation of

ENaC, to which Nedd4-2 recognizes and induces the ubiquitination of surface ENaC only without affecting immature ENaC within the cytoplasm (Zhou et al., 2007). This phenomenon can be explained by the existence of the C2 domain in Nedd4-2, which could recruit Nedd4-2 to the plasma membrane by interacting with phospholipids of the cell membrane (Nalefski & Falke,

1996; Plant et al., 2000). In addition, our recent work also showed that the integral membrane protein caveolin-3 recruits Nedd4-2 from the cytosol to the plasma membrane (Guo et al., 2012), thus, making it likely that Nedd4-2 accumulates around the cell membrane and preferentially interacts with membrane-localized mature hERG channels (Figure 25).

As previously mentioned, I proposed that ubiquitination of Nedd4-2 takes place at the plasma membrane after it interacts with and mediates ubiquitination of hERG channels. It is likely that ubiquitinated Nedd4-2 can be either degraded or recycled back to the plasma membrane via early endosomes (Figure 25). Rab4 is well known to be associated with early endosomes mediating the

rapid recycling of internalized proteins back to the plasma membrane (Seachrist et al., 2000; Li et al., 2008). Thus, while Nedd4-2 is ubiquitinated and undergoes endocytosis, Rab4 may facilitate the recycling of Nedd4-2 at the early stages along the degradation pathway and lead to decreased degradation and increased expression. The increased Nedd4-2 can be recruited to the plasma membrane, consequently leading to an enhanced degradation of mature hERG channels (Figure

25).

It is a novel finding that Rab4 regulates the expression of mature hERG on the plasma membrane via enhancing the expression of Nedd4-2. Unlike Rab4-mediated recycling of the

Angiotensin II type 1 receptor and the β-adrenergic receptor, the direct effect of Rab4 on the recycling of internalized hERG channels is negligible as revealed by the Nedd4-2 insensitive mutant hERG channels ∆1073 and Y1078A. Interestingly, the effect of Rab4 on the recycling of

Nedd4-2 is robust, causing further degradation of hERG on the plasma membrane. These findings may have broad applications to other Nedd4-2 substrates that possess the PY motif. For example,

Saxena et al. (2006a; 2006b) demonstrated that Rab4 overexpression decreased the plasma membrane-expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR). However, the mechanism of how Rab4 regulates these two channels remains unclear. It has only been suggested that ENaC incorporates Rab GTPase as an essential element for channel function, and Rab4 regulates the expression of ENaC in the apical membrane of epithelial cells (Saxena et al., 2006b). It is likely that Rab4 also regulates these two channels via Nedd4-2, because both ENaC and CFTR are Nedd4-2 substrates (Debonneville et al.,

2001; Zhou et al., 2007; Caohuy et al., 2009). In addition, cardiac and neuronal Na+ channels, the cardiac K+ channel KCNQ1, and neuronal K+ channels KCNQ2/3/5 are among the substrates of

Nedd4-2 (Fotia et al., 2004; van Bemmelen et al., 2004; Ekberg et al., 2007; Jespersen et al.,

2007). Besides the direct effects on these channels, Rab4 is expected to regulate these channels via altered Nedd4-2 expression levels. Finally, Nedd4-2 is involved in a variety of cellular processes, including neuronal development and cell growth (Morén et al., 2005; Yang & Kumar,

2009). As such, it has been demonstrated that the Rab4-mediated regulatory mechanism of

Nedd4-2 may have significant impacts on various cellular processes.

Rab4 expression is highly variable and regulated in cardiomyocytes (Wu et al., 2001; Odley et al., 2004; Etzion et al., 2010). Our data demonstrated that Rab4-mediated alterations in ERG expression also exist in cardiomyocytes (Figure 22). The Rab4-mediated down-regulation of mature hERG expression may underlie the mechanisms of cardiomyopathic changes in LQTS patients. Haugaa et al. (2012) reported that almost 20% of LQTS patients develop subclinical cardiomyopathic changes, including increased left atrial volume indices and ventricular enlargement. It has been reported that patients with cardiomyopathy, such as Takotsubo

Cardiomyopathy (TCM), also develop prolonged QT interval, increasing the risk of sudden death within affected individuals (Behr & Mahida, 2009). A previous study has demonstrated that cardiac Rab4 is up-regulated in a dilated cardiomyopathy model overexpressing β2-adrenergic receptors (Wu et al., 2001). The up-regulation of Rab4 expression is also observed in ventricular tissues of Akt2-knockout mice, which develop a syndrome similar to diabetes mellitus type 2 cardiomyopathy (Etzion et al., 2010). Transgenic overexpression of Rab4 in the mouse myocardium induces concentric cardiac hypertrophy (Filipeanu et al., 2006). Cardiomyopathy, as well as heart failure is closely associated with QT prolongation and ventricular arrhythmias

(Martin et al., 1994; Marbán, 1999a; Tomaselli & Marbán, 1999; Johnson et al., 2011; Madias et al., 2011). Specifically, a link between depressed hERG channel function and abnormal QT prolongation in diabetic rabbits was also reported (Zhang, 2006). The relationship between the

expression of Rab4 and hERG channels is worth investigating in LQTS animal model. Thus, our study raised the possibility that elevated Rab4-mediated reduction in hERG expression may play a role in the development of QT prolongation in patients with cardiomyopathy and heart failure.

In summary, the present study has revealed a novel mechanism for the Rab4-mediated regulation of Nedd4-2 and hERG channels, in that Rab4 increases Nedd4-2 expression via recycling which subsequently decreases the expression of mature hERG channels. Rab4-mediated

Nedd4-2 regulation may potentially impact other cellular processes also regulated by Nedd4-2.

Future Directions

The signaling mechanism of the recycling process is still not fully understood. Within a pool of molecules destined for degradation, only a fraction of molecules are signaled for recycling at the early stage, while the remainder are targeted for degradation. The identity of the up- and downstream adaptors and effectors that are involved in Rab4-mediated recycling at the early endosome still requires further investigation.

In addition, as ubiquitinated Nedd4-2 is recycled by Rab4, deubiquitination should also occur, allowing recycled protein to be functional again. Deubiquitination is mediated by deubiquitinating enzymes (DUBs), and is involved in various cellular processes, such as processing ubiquitin precursors and freeing proteasomes. The DUBs that are involved in the

Rab4-mediated deubiquitination are not well understood. It would be worth investigating these

DUBs to potentially develop some therapeutic interventions.

As mentioned in the discussion, the function of Rab4 in cardiomyocytes is highly variable and regulated. As cardiomyopathy and heart failure are closely associated with cardiac arrhythmias, and increased expression of endogenous Rab4 is observed in a rat model overexpressing β2-

adrenergic receptors as well as in a Akt2-/- mice model, an animal model investigating the effect of Rab4 on hERG channels in vivo is of great interest to understand the relationship between cardiomyopathy and LQTS.

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