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Theses & Dissertations
Boston University Theses & Dissertations
2014
Infant sudden death: a novel
mutation responsible for impaired
sodium channel function
Morganstein, Jace Grant
http://hdl.handle.net/2144/15047
Boston University
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BOSTON UNIVERSITY
SCHOOL OF MEDICINE
Thesis
INFANT SUDDEN DEATH: A NOVEL MUTATION RESPONSIBLE FOR
IMPAIRED SODIUM CHANNEL FUNCTION
by
JACE MORGANSTEIN
B.S., University of Michigan, 2012
Submitted in partial fulfillment of the
requirements for the degree of
Master of Science
2014
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© 2014 by
JACE MORGANSTEIN
All rights reserved
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Approved by
First Reader
Simon Levy, Ph.D.
Associate Professor of Physiology and Biophysics
Second Reader
William Coetzee, D.Sc.
Professor; Director of Research
New York University Langone Medical Center
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DEDICATION
I would like to dedicate this work to my mom, dad, brother, and Brandon
McCartney.
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ACKNOWLEDGMENTS
I wish to express my deepest gratitude to William Coetzee, D.Sc., Kundan Jana,
M.D., Monique Foster, B.S., and Joanne Ho, B.S. You all taught me so much and
made my experience in Coetzee Lab a truly enriching one.
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INFANT SUDDEN DEATH: A NOVEL MUTATION RESPONSIBLE FOR
IMPAIRED SODIUM CHANNEL FUNCTION
JACE MORGANSTEIN
ABSTRACT
In coordination with the New York City Medical Examiner’s Office, we
received the sequence of a mutated SCN5A gene that was found in a five-weekold girl who died in her sleep. SCN5A codes for the voltage-gated cardiac sodium
channel alpha subunit (Nav1.5) and is responsible for the fast depolarization in
phase zero of the cardiac action potential. The mutations that were present in the
girl’s SCN5A gene were a missense mutation, Q1832E, and a truncation
mutation, R1944X. In order to gain an understanding of the conditions that led to
the patient’s death, we carried out a functional analysis on the mutant channels
and measured how their properties differed from wild type Nav1.5 properties.!
For our functional analysis we carried out mutagenesis reactions to produce
three experimental constructs in order to examine independent effects of
Q1832E or R1944X, and to examine their interaction (mutant Nav1.5 that
contains both Q1832E or R1944X; as was found in the genetic screen). These
constructs were transfected into HEK 293 cells and studied using the patch
clamp analysis using the whole cell configuration. Experiments were carried out
to test the Nav1.5 current voltage relationships, the recovery from inactivation
properties, and steady state inactivation properties.!
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The data demonstrated that each of the three constructs resulted in a
significantly reduced current density when compared to wild type Nav1.5
currents. The gating properties of the mutant channels were similar to those of
wild type Nav1.5, though Nav1.5-R1944X did show a statistically significant
slower recovery from inactivation than the wild type channel. Though more
experimentation is needed to determine the mechanism behind the reduced
current in the mutant channels, our data shows that each of the mutations is
sufficient to produce a severely dysfunctional channel and this is likely the cause
of the patient’s death.
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TABLE OF CONTENTS
TITLE!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!...i
COPYRIGHT PAGE!!!!!!!!!!!!!!!!!..!!!!!!!!...ii
READER APPROVAL PAGE!!!!!!!!!!!!!!!!!!!!...!..iii
DEDICATION ....................................................................................................... iv
ACKNOWLEDGMENTS ........................................................................................ v
ABSTRACT .......................................................................................................... vi
TABLE OF CONTENTS ...................................................................................... viii
LIST OF TABLES .................................................................................................. x
LIST OF FIGURES ............................................................................................... xi
LIST OF ABBREVIATIONS ..................................................................................xii
INTRODUCTION ................................................................................................... 1
The Cardiac Conduction System ............................................................ 1
The Electrocardiogram!!!!!!!!!!!!!!!!......!!!...2
The Cardiac Action Potential!!!!!!!!!!!!!!....!!!..5
The SCN5A gene !!!!!!........!!!!!!!!!!!!!!!...8
Channelopaties of SCN5A!!!!!!!!!!!!!.!!!!!!10
Specific Aims...........................................................................................12
METHODS........................................................................................................... 14
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Preparation of Plasmid Constructs!!!!!!!!!!!!!!!.14
Mutagenesis.!!!!!....!!!!!!!!!!!!!!!!!!!.14
Cell Culture !!!!!!!!!!!!!!!...!!!!!....!!!...15
Patch Clamp Analysis !!!!!!!!!!...!!!!....!!!!!16
RESULTS ............................................................................................................ 19
Effects on Current Amplitude.!!!!!!!!!!..!!!!!!!19
Time Constant of Inactivation.!!!!!!!!!!..!!!!!!!23
Recovery from Inactivation.!!!!!!!!!!!.!!!!!!!.24
Steady State Inactivation.!!!!!!!!!!!!!!!!!!!..27
DISCUSSION!!!!!!!!!!!!!!!!!!!!!!!!!!!!..30
Q1832E.!!!!!!!!!!!!!!!!!!..!!!!!!!!!.30
R1944X.!!!!!!!!!!...!!!!!!!!!!!!!!!!!31
Double Mutation.!!!!!!!!!!..!!!!!!!!!!!!!32
Clinical Correlations!.!!!!!!......!!!!!!!!!!!!!32
Limitations and Future Studies.!!!!!..!!........!!!!!!!33
REFERENCES .................................................................................................... 36
CURRICULUM VITAE ......................................................................................... 43
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LIST OF TABLES
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Table
Title
Page
1
Time Constants of Inactivation
24
2
Recovery from Inactivation Time Constants
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x
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LIST OF FIGURES
Figure
Title
Page
1
The Cardiac Action Potential
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Current Voltage Relationships for Wild Type and
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Mutant Channels
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Time Courses for Recovery from Inactivation
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Steady State Inactivation
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xi
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LIST OF ABBREVIATIONS
AV .................................................................................................... Atrioventricular
Ca2+ ............................................................................................................ Calcium
CaCl2 ............................................................................................ Calcium Chloride
cDNA .......................................................... Complimentary Deoxyribonucleic Acid
cm ......................................................................................................... Centimeters
CMV.............................................................................................. Cytomegalovirus
CsF ................................................................................................ Cesium Fluoride
CsOH .......................................................................................... Cesium Hydroxide
DNA ...................................................................................... Deoxyribonucleic Acid
E. Coli ............................................................................................ Escherichia Coli
EGTA ................................................................... Ethylene Glycol Tetraacetic Acid
ECG ............................................................................................ Electrocardiogram
FBS.......................................................................................... Fetal Bovine Serum
GFP ............................................................................... Green Fluorescent Protein
GST ............................................................................... Glutathione S-Transferase
G" ............................................................................................................ Gigaohm
HEK Cells ............................................................. Human Embryonic Kidney Cells
Ip ........................................................................................................ Patch Current
IV .................................................................................................... Current-Voltage
K+ ............................................................................................................ Potassium
KCl ............................................................................................ Potassium Chloride
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LQTS ........................................................................................ Long QT Syndrome
LQT3............................................................................. Long QT Syndrome Type 3
MEM ............................................................................ Minimum Essential Medium
MgCl2 ...................................................................................... Magnesium Chloride
ms .......................................................................................................... Millisecond
mV ............................................................................................................... Millivolt
M" ........................................................................................................... Megaohm
Na+............................................................................................................... Sodium
NaCl.............................................................................................. Sodium Chloride
NaOH.......................................................................................... Sodium Hydroxide
Nav1.5 ............................................................. Voltage Gated Sodium Channel 1.5
pA/pF .................................................................................. Picoamp Per Picofarad
PCR ............................................................................ Polymerase Chain Reaction
PTPH1 ............................................................... Protein Tyrosine Phosphatase H1
SAP97 ................................................................... Synapse-Associated Protein 97
TEA-Cl ..................................................................... Tetraethylammonium Chloride
Rf ............................................................................................... Feedback Resistor
SA ............................................................................................................. Sinoatrial
SCC ............................................................................... Sodium Channel Complex
SCN5A............................................. Gene Encoding the Cardiac Sodium Channel
SEM ............................................................................. Standard Error of the Mean
SIDS ...................................................................... Sudden Infant Death Syndrome
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V .................................................................................................................. Voltage
WT ........................................................................................................... Wild Type
µF ........................................................................................................... Microfarad
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INTRODUCTION
The Cardiac Conduction System
The heart is comprised of three layers of tissue: the epicardium, the
myocardium and the endocardium. The myocardium is made up solely of cardiac
muscle, a type of striated muscle unique to the heart (Iaizzo, 2009). The basic
units of cardiac muscle are the muscle cells, or the cardiomyocytes, which are
connected by intercalated discs — structures rich in gap junctions that allow for
electrical conduction between cardiomyocytes and hence the synchronous
contraction of the tissue upon excitation by an electrical stimulus (Franke,!
Borrmann, Grund, & Pieperhoff, 2006).
The electrical impulse that initiates the heart beat is generated in the
sinoatrial (SA) node, a crescent shaped structure located in the wall of the right
atrium (Anderson, Ho, & Anderson, 1979). It is then propagated through the
cardiomyocytes of the atria, causing a contraction in these chambers, which is
responsible for filling of the ventricles with blood. The electrical signal then
reaches the atrioventricular (AV) node, situated at the base of the atrial septum,
where a delay in propagation occurs. This delay ensures complete filling of the
ventricles before they contract. The electrical impulse is then rapidly propagated
through the cardiac conduction system to excite the ventricles, such that
contraction starts from the apex of the heart and spreads to the base. This
contraction sequence ensures optimal expulsion of blood into the aorta, from
which blood will flow to somatic tissues.!
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The Electrocardiogram!
The propagation of the electrical impulse through the heart can be
measured by an electrocardiogram, or ECG. The ECG is a fundamental clinical
tool used to observe heart rhythm and to detect diseases of cardiac
electrophysiology. To record an ECG, electrodes are placed on the chest wall
and limbs, and electrical readings are transcribed to standardized graph paper or
another recording device. Typically, 12 electrodes are used; six on the chest and
six on the limbs (Morris, Brady, & Camm, 2008). The waveforms generated
correspond to the electrical activity of the heart. The shape and amplitude of the
waveforms are determined by the direction of the movement of the electrical
signals in relation to the electrodes. The direction of the deflections depends on
whether the impulse is moving toward or away from the recording electrodes—an
impulse moving toward an electrode produces a positive deflection, and one
moving away from an electrode produces a deflection in the negative direction
(Ellis, 2011). The amplitude of the deflections is influenced by the size of the
section myocardium the impulse is moving through, the properties of the tissue
between the heart and the electrodes, and the distance between the electrode
and the myocardium (Schmidt, 1963).!
In order to properly interpret an ECG and identify any abnormalities in the
recording, the individual components of the normal waveform must be
understood. Firing of the SA node and subsequent depolarization of the atria
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produces the first upward deflection of the ECG waveform, the P wave. Since the
atria have relatively low muscle mass and because they depolarize almost
simultaneously, a single, small upward deflection is produced (Morris et al.,!
2008). The PR interval is the brief return to baseline after the P wave,
corresponding to the time between atrial contraction and ventricular
depolarization. The most prominent wave in the normal ECG is the QRS
complex. Corresponding to ventricular depolarization, the QRS complex contains
three portions: a downward Q wave followed by an upward deflecting R wave
and a terminal downward deflecting S wave. Depolarization of the left ventricle
dominates the reading on the ECG because its myocardial mass is larger than
that of the right ventricle. The subsequent flat segment of the ECG waveform is
the ST segment, which begins at the J point—the point at which the QRS
complex terminates (Ellis, 2011). The T wave is the feature following the ST
segment. It is upright and asymmetrical on the normal ECG, and precedes the U
wave, which is the final feature of the normal ECG waveform. The U wave is the
recording caused by the repolarization of the mid-myocardial cells in the
ventricles (Pérez Riera et al., 2008).!
There are many cardiac abnormalities that can be observed and
diagnosed by evaluation of an ECG recording. First, abnormal heart rates can be
detected by an ECG by measuring the amount of time between R waves;
tachycardia is defined as a rate greater than 100 beats per minute and
bradycardia is the presence of a heart rate slower than 60 beats per minute. Sick
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Sinus Syndrome is defined as severe sinus bradycardia, and results from
dysfunction of the SA node (Morris et al., 2008).!
Electrical abnormalities pertaining to the atria that can be detected by an!
ECG include atrial fibrillation and atrial tachycardia. Atrial fibrillation is caused by
multiple waves of activation propagating across the atrium, generated by rapidly
firing foci. It presents on the ECG with a wavy baseline and an absent P wave.!
Atrial tachycardia is caused by an aberrant source of electrical impulse in the
atrium, resulting in abnormally shaped P waves (Simpson, Foster, Woelfel, &!
Gettes, 1986).!
Atrioventricular conduction blocks can also be observed on the ECG. AV
blocks occur when electrical impulses are not properly conducted from the atria
to the ventricles, and can be categorized into three discreet groups based on
how they present on the ECG. A first degree AV block occurs when there is a
delay in the AV node’s propagation of the electrical impulse from the atria to the
ventricles, presenting as a prolonged PR interval on the ECG. A second degree
AV block is characterized by intermittent failure of the AV node to propagate the
electrical impulse from the atria to the ventricles. When this occurs, some P
waves are not followed by QRS complexes on the ECG recording. A third degree!
AV block occurs when there is complete failure of conduction between the atria
and ventricles, which contract independently (“Atrioventricular block, ECG
tracing,” n.d.).
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Blockages of either the right or left branch of the bundle of His can also be
detected by an ECG. In right bundle branch block, electrical impulses travelling
through the left bundle branch cause a normal contraction of the left ventricle.!
This wave of depolarization is spread through the non-specialized myocardium to
the right ventricle, resulting in a delayed contraction of this chamber (Morris et
al., 2008). On the ECG, this phenomenon presents as a delayed and abnormally
pronounced R wave in the right precordial leads and a terminal S wave in the left
precordial leads. In left bundle branch block, septal Q waves are replaced with R
waves and QRS complexes are elongated (Cecil, Goldman, & Bennett, 2000).!
Ventricular tachycardia is an abnormally fast heart rate originating in the
ventricles. This presents on the ECG as large deflections that indicate a heart
rate faster than 100 beats per minute. Sometimes, ventricular tachycardia can
deteriorate into ventricular fibrillation, characterized by a heart rate of greater
than 300 beats per minute that has lost all normal coordination (Tung, Boyle, &
Shivkumar, 2010). Torsades de pointes is a ventricular tachycardia characterized
by a rotating in the cardiac axis and a similarly changing QRS complex. Torsades
de pointes is associated with long QT syndrome (Yap & Camm, 2003).
The Cardiac Action Potential
The cardiac action potential is a cellular event and the propagated action
potential represents the electrical impulse that sweeps across the heart, from cell
to cell, during contraction. As shown in Figure 1, the action potential is often
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characterized as having five discreet phases, beginning with phase zero, the
rapid depolarization of the cardiomyocyte. Rapid depolarization of the heart
muscle cells results primarily from the opening of voltage gated Na+ channels,
most significantly the Nav1.5 channels encoded by the SCN5A gene (Patlak &
Ortiz, 1985). During this phase the membrane depolarizes to approximately +40
mV (Nattel & Carlsson, 2006).
Early partial repolarization follows the rapid depolarization that initiates the
cardiac action potential (Figure 1). Approximately one to two milliseconds after
they are activated, the voltage gated sodium channels inactivate and transient
outward potassium channels are activated (Rook et al., 1999). This repolarizes
the membrane potential to approximately +10 mV (phase 1 of the action
potential) and normally occurs within 50 milliseconds after the action potential
was initiated (Nattel & Carlsson, 2006).
Perhaps the most distinct feature of the cardiac action potential is the
plateau phase, or phase 2 (Figure 1). In this phase, L-type Ca2+ channels, which
were activated several milliseconds after the initial rapid depolarization, open for
approximately 200 milliseconds and keep the membrane depolarized relative to
resting potential (Pinnell, Turner, & Howell, 2007). The influx of Ca2+ that results
from the opening of these channels is the trigger for contraction of the myocyte
(Roderick, Berridge, & Bootman, 2003). During the plateau phase, delayed
rectifying K+ channels gradually open, causing the cell to repolarize (Pinnell et
al., 2007).
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Phase three of the cardiac action potential is rapid repolarization of the
membrane potential back to negative “resting” levels (Figure 1). As the amount of
current passing through the delayed rectifying potassium channels increases—
especially via the IKs current—the L-type calcium channels begin to close. This
results in a terminal repolarization of the membrane potential, quickened by the
IK1 current (Jost et al., 2009).
The final phase of the cardiac action potential is phase four, the resting
phase (Figure 1). At this phase the prominent current is IK1, and the resting
potential is approximately -90 mV (Jost et al., 2009).
Figure 1: The Cardiac Action Potential. A trace of the cardiac action potential is
shown, with each of the discreet phases labeled with their corresponding number.
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The SCN5A gene
The gene SCN5A codes for the #-subunit of the cardiac Na+ channel,
Nav1.5, also sometimes referred to as SCN5A (in uppercase, and non-italicized
to distinguish it from the SCN5A gene). Nav1.5 is one of nine isoforms of the
voltage-dependent Na+ channel subunits identified in mammals. The Nav1.5
subunit consists of four homologous domains, each with six alpha helical
transmembrane segments, denoted as S1 to S6. The S4 segments act as the
voltage sensor and segments S5 and S6 of each domain form the pore lining
helices (Catterall, 2000).
When activated by a depolarizing stimulus, the Nav1.5 channel remains
open for only a few milliseconds before it spontaneously inactivates. The
inactivation gate responsible for this phenomenon is located in the intracellular
loop connecting domains III and IV (Benzanilla et al., 1987). The hydrophobic
triad of isoleucine, phenylalanine and methionine amino acid residues is required
for fast inactivation. In fact, peptides containing this motif can act as Nav1.5
blockers (S.-Y. Wang & Wang, 2005). Phenylalanine F1489 projects out from the
inactivation gate, in a position that is ideal for occlusion of the pore. The
methionine of the motif interacts with two tyrosine residues in an alpha helix to
which the motif is tethered. This hydrophobic interaction stabilizes the orientation
of the peptide that forces F1489 into its exposed position (Kellenberger, West,
Catterall, & Scheuer, 1997). The inactivation of SCN5A derives its voltage
dependence from coupling to the activation process driven by the S4 voltage
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sensor in domain IV (Catterall, 2000). Nav1.5 subunits associate in large protein
complexes called the Sodium Channel Complex (SCC). The proteins that make
up the SCC regulate Na+ channel function and Na+ channel subunit localization
(Adsit, Vaidyanathan, Galler, Kyle, & Makielski, 2013). The 243-residue
intracellular C terminal section of Nav1.5 contains several protein interaction
motifs that aid in the formation of the SCC. The three most well studied and
identified are the IQ motif, the PY motif and the PDZ binding motif (Shy, Gillet, &
Abriel, 2013).
PDZ motifs are conserved domains that interact with C terminal ends of
target proteins. The terminal three residues of Nav1.5 —serine, isoleucine, and
valine—constitute a motif that interacts with PDZ domain binding proteins. Such
proteins include PTPH1, SAP97 and syntrophins (“The PDZ Domain as a
Complex Adaptive System,” n.d.). Through GST pull down experiments,
syntrophins have been shown to interact with the C terminus of Nav1.5. It has
been demonstrated that when syntrophins interact with the PDZ domain of
Nav1.5, the steady state activation of the Na+ current is shifted (Ou et al., 2003).
The interaction between Nav1.5 and PTPH1 at the PDZ binding domain has been
shown to shift the availability of the channels to hyperpolarized potentials
(Jespersen et al., 2006).
The scaffolding protein SAP97 also interacts with the PDZ binding domain
motif, and cells with silenced SAP97 showed a similar reduction of current as
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Nav1.5 subunits that lack the final three amino acids of the C terminal (Petitprez
et al., 2011).
Another protein association motif near the C terminus of Nav1.5 is the PYmotif. Here, E3 ubiquitin-protein ligases bind, allowing for the ion channel to be
regulated via ubiquitination. This type of regulation is a post-translational
modification that signals for target proteins to be degraded or transported to other
membrane compartments (Remme, 2013).
The IQ motif near the C terminus of Nav1.5 constitutes a calmodulin
binding motif. Calmodulin has been shown to directly interact with Nav1.5,
however, there have been many inconsistent functional consequences of this
interaction reported (Shy et al., 2013).
Other protein interactions at the C terminus have been observed without
the identification of a specific binding motif. For example, fibroblast growth factor
homologous family members have been shown to interact with a region of the
Nav1.5 C terminus from amino acid residues 1773 to 1832 (Liu, Dib-Hajj,
Renganathan, Cummins, & Waxman, 2003).
Channelopathies of SCN5A
Long QT Syndrome (LQTS) is an inherited arrhythmogenic disease that is
characterized by elongation of the QT interval on the ECG. When the elongation
in the QT interval is due to a mutation of SCN5A, the disease is known as LQT3
(Mearns, 2010). To date, there have been more than 80 SCN5A mutations
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reported to cause LQT3 (Maugeri, n.d.). The first SCN5A mutation characterized
was a mutation causing deletion of amino acid residues 1505 to 1507 of Nav1.5.
Named $KPQ, this mutation was shown to cause an abnormal sustained Na +
current that did not decay during a 200 ms depolarizing pulse of -20 mV
(Bennett, Yazawa, Makita, & George, 1995). This maintenance of Na+ current for
a period longer than what is seen in the wild type (WT) has been shown in most
other SCN5A mutations linked to the LQT3 syndrome. Called late Na+ current or
persistent Na+ current, this feature is considered to be the major pathogenic
feature of LQT3 (P. Wang, Hsia, Al-Ahmad, & Zei, 2009). All mutations linked to
LQT3 can be characterized as gain-of-function mutations, as they increase the
sodium current over the voltage range or time course relative to wild type
channels. Gain-of-function mutations disturb the balance between inward and
outward current during the plateau phase of the action potential and prolong
repolarization (D. W. Wang, Yazawa, Makita, George, & Bennett, 1997).
Brugada syndrome, another disease of Nav1.5 channels, is characterized
by ST segment elevation in the right precordial leads of the ECG. Often a distinct
J-wave on the ECG is observed in people with Brugada syndrome (Grant, 2009).
This disease can cause sudden cardiac death resulting from episodes of
ventricular tachycardia. Brugada syndrome has also been extensively studied, as
there have been over 90 SCN5A mutations identified that result in the disease
(Maugeri, n.d.).
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Unlike LQT3, mutations that result in Brugada Syndrome are always lossof-function mutations. The common underlying mechanism of Brugada Syndrome
is reduced current density, which can be due to abnormal trafficking or a diverse
array of biophysical changes to the channel (Baroudi et al., 2001).
Specific Aims
Through correspondence with the New York City Medical Examiner, we
received the sequence of a mutated SCN5A gene that was present in a fiveweek-old female who died suddenly in her sleep. The gene contained two
separate mutations, a missense mutation at nucleotide 5504, changing the
normally present cytosine to a guanine, and a nonsense mutation at 5830,
changing the normally present cytosine to a thymine. The missense mutation
results in the substitution of Glutamine at amino acid residue 1832 for a
Glutamate residue of Nav1.5. The nonsense mutation results in a point mutation
that introduces a stop codon at amino acid position 1944, resulting in the
truncation of the C terminus. While the truncation mutation, R1944X, has never
been described, the missense mutation Q1832E has previously been identified
as a mutation that results in Brugada Syndrome (Kapplinger et al., 2010).
However, Kapplinger et al. did not carry out a functional analysis of the mutated
channel, but instead identified its presence in a patient who died suddenly from
Brugada Syndrome. It was our goal to carry out a functional analysis of these
mutated Nav1.5 channels in order to shed light on the conditions that lead to the
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death of the five week old victim. The functional analysis was carried out using
the patch clamp technique in the whole cell configuration to determine if these
mutant channels behave differently from wild type Nav1.5 channels.
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METHODS
Preparation of Plasmid Constructs
The Nav1.5 cDNA was received in a pcDNA3.1 vector as a generous gift
from Nicole Schmitt, Ph.D., University of Copenhagen. This construct was
appropriate for our use, as pcDNA3.1 is a vector with the CMV promoter, suitable
for expression in mammalian cells, for example when transfected into HEK cells.
The cDNA was propagated in and purified from an E.Coli bacterial culture using
the Quantum Prep Plasmid Midiprep Kit (Bio Rad, Catalog #732-6120, Hercules,
CA).
Mutagenesis
Using the wild type plasmid as template, three mutant constructs were
created: a construct containing Nav1.5 with only the Q1832E mutation, a
construct containing the early C-terminal truncation (R1944X), and a construct
containing both mutations. Custom primers were designed for the site-directed
mutagenesis, which contained the mutation of interest, flanked on either side by
approximately 10 nucleotides of wild type. Mutations were made using the
Quickchange II XL Site Directed Mutagenesis Kit (Aligent Technologies, Catalog
#200521, Santa Clara, CA). The mutagenic primers were hybridized to their
complementary sequence in single-stranded wild type DNA, forming a
heteroduplex with mismatched nucleotides at the site of the mutation. A
polymerase chain reaction (PCR) was then carried out in which a Pfu polymerase
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enzyme converted the single stranded DNA into double-stranded form, using the
wild type strand as a template. When this process was completed, all regions of
the plasmid except the region containing the mutagenic primer were wild type,
forming a heteroduplex cDNA in which one strand contained the wild type
sequence and the other contained the mutant sequence. Continuation of the
PCR amplified the mutant plasmid, and this PCR product was transformed into
E.Coli. The bacteria were then plated on a petri dish containing agar media and
ampicillin, for which the construct contained an antibiotic resistant gene. This
allowed for the selective growth of colonies that contained the mutated plasmid.
The colonies were then propagated, lysed to extract cDNA and the plasmids
were purified (QIAPrep Spin Miniprep protocol, Qiagen, Catalog #27104, Venlo,
Limburg). To verify successful acquisition of the mutated DNA, plasmids were
sequenced (Macrogen USA, Rockville, MD).
Cell Culture
For functional analysis, it was necessary to choose a cell type that could
be efficiently transfected and was appropriate for study using the patch clamp
technique. The cells chosen were Human Embryonic Kidney 293 cells, or HEK293 cells, which exhibit high efficiency of transfection and protein production,
faithful translation and processing of proteins, and small cell size with minimal
processes— features that are ideal for patch clamp experiments.
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The HEK 293 cells were cultured in Corning’s Minimum Essential Medium
(MEM) (Corning, Catalog #10-009, Manassas, VA), supplemented with 10%
Fetal Bovine Serum (FBS) and Penicillin-Streptomycin. Cells were grown on
poly-lysine coated coverslips in 16mm petri dishes until 70-80% confluence was
reached. Media was then replaced with an antibiotic-free mixture of MEM and
10% FBS before transfection using the Lipofectamine 2000 reagent (Life
Technologies, catalog #1668-019, Carlsbad, CA). For the transfections, 2%g of
DNA was used—1.5 %g of the construct to be studied and 0.5 %g of GFP or
mCherry plasmids, so that the successfully transfected cells could be identified
for patch clamping. Cells were then incubated overnight and used for patch
clamp analysis the next day.
Patch Clamp Analysis
To study how currents produced from the mutant Nav1.5 currents differed
from wild type Nav1.5 current, the patch clamp technique was employed. In this
technique, a patch pipette is connected to the inverting input of a feedback
amplifier. Because the input resistance of this amplifier is essentially infinite, all of
the current recorded by the pipette flows through a feedback resistor Rf,
connected in parallel to the feedback amplifier. The feedback resistor has very
high resistance, typically 10G",allowing for the measurement of tiny currents —
as the patch current (Ip) is given by the voltage drop across the feedback resistor,
i.e., V=IpRf. The command potential is applied to another input of the feedback
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amplifier and the amplifier passes current through the feedback resistor to keep
the voltage at the inverting input the same as the command voltage. This allows
for the desired potential to also be applied to the pipette, and therefore the cell
being studied (Ashcroft, 2000).
In our experiments, patch pipettes of resistances between 2-4 M" were
pulled from borosilicate glass using a Zietz DMZ universal puller (Zietz
Instruments, Martinsried, Germany). The pipettes were then coated with
Sigmacote (Sigma-Aldrich Co., catalog #SL2-25ML, St. Louis, MO) to increase
their hydrophobicity. Using the Burleigh PCS-6000 micromanipulator (Thorlabs,
Newton, NJ), pipettes were positioned onto the cell membrane of an isolated
fluorescent cell and suction was applied to form a high resistance seal of around
10G" between the cell membrane and the glass w all of the micropipette. The
high resistance of this seal allowed for the recording of currents with very little
background noise and also for the membrane potential of the cell to be altered by
applying a voltage to the pipette. As we were fundamentally interested in
measuring activity of all of the ion channels in the cell membrane, we employed
the whole-cell technique. Upon formation of the gigaseal, additional suction was
applied to destroy the section of membrane attached to the pipette and provide
electrical access to the cell interior. This allowed for the measurement of the
whole cell current upon application of a voltage to the pipette.
Data was acquired using the Axopatch 200B patch clamp amplifier and
pClamp 10.2 software (Axon Instruments, Sunnyvale, CA). Pipette resistance
17
!
and junction potential were corrected and whole-cell capacitance and series
resistance were compensated to levels greater than 80%. The extracellular bath
solution used was a standard Tyrode’s solution, containing (in mmol/L): 137
NaCl, 5.4 KCl, 10 HEPES, 0.5 MgCl2, 1.8 CaCl2, 10 glucose and was pH
adjusted to 7.4 using 2N NaOH. The intracellular pipette solution contained (in
mmol/L): 10 KCl, 105 CsF, 10 NaCl, 10 HEPES, 10 EGTA, and 10 TEA-Cl and
was pH adjusted to 7.2 using 2N CsOH.
Data from all experiments were analyzed using pClamp 10.2 software and
plotted using OriginPro 8 (OriginLab, Northampton, MA). Results are presented
as mean ± standard error of the mean. Statistical comparisons were performed
using an unpaired Student’s t-test and statistical significance was assumed when
p < 0.05.
18
!
RESULTS
Effects on current amplitude
In order to determine how the channels were functionally affected by the
mutations, we carried out experiments to analyze the current-voltage
relationships for the mutant and wild type channels. In experiments examining
the current-voltage relationship for a particular channel, voltages are applied to
the membrane, changing the membrane potential, and the currents elicited from
these various potentials is recorded. If the mutations had no effect on the
channel’s function, identical current voltage curves would be expected.
In order to test the current voltage relationship for our channels, the
voltage protocol depicted in Figure 2A was applied. As the Nav1.5 channel is
voltage gated and opens at depolarized membrane potentials, the range chosen
for test voltages was -80 mV to 60 mV—a range in which no channels should be
open at the most negative potentials, then the maximum amount of channels
should be open at the least negative potentials. In this protocol, the increasingly
depolarizing voltages elicited larger currents until the command potential reached
-20mV, after which point smaller currents were produced. This is because the
current elicited from a test voltage depends on the number of channels that are
open as well as the electrochemical driving force for ionic movement across the
membrane. Therefore, while more positive potentials opened a greater proportion
of channels, they reduced the driving force for the positive sodium ion to enter
the cell. At -20 mV there is a significant driving force to cause maximal ion flux
19
!
through the channels. By convention an inward flux of positive ions is shown as a
negative deflection in the trace.
20
!
A
B
Figure 2: Current Voltage Relationships for Wild Type and Mutant Channels
A) The voltage protocol used to measure current voltage relationships for each channel.
The holding potential was set to -120 mV and voltage pulses ranging from -80 mV to 60
mV were applied, stepping up in 10 mV increments.
B) Current voltage relationships for each channel being studies. Current was normalized
to cell capacitance to get a measure of current density.
21
!
To visualize the relationship of the current density elicited for each test
voltage, an I-V curve was produced, as shown in Figure 2B. To produce this
graph, the maximum current for each trace was recorded and plotted against
the corresponding potential for which it was elicited. As the amount of current
produced varies with cell size, the value for the maximum current was
normalized against the value for the cell capacitance, which is directly
correlated to cell size (the specific membrane capacitance is 1%F/ cm-2). This
produces a graph that has mV units on the x-axis and pA/pF units on the y-axis.
As can be seen in Figure 2B, the amount of current produced for each of the
mutated channels is greatly reduced when compared to currents from wild type
channels. In fact, wild type channels produced a maximum current at –20 mV of 328.47 pA/pF, whereas channels with the missense mutation produced maximal
currents at –20 mV of -57.9 pA/pF. Channels containing the truncation mutation
R1944X alone also showed greatly reduced current, with the maximum current
elicited being -86.6 pA/pF. Curiously, those channels containing both the
missense and the truncation mutation produced greater currents than channels
containing either of the two mutations alone. However, this difference was not
statistically significant and the double mutant channels also produced currents
that were drastically smaller than wild type currents. The maximum current
produced from the channels carrying both mutations was -102.3 pA/pF. The
current densities of the Q1832E mutant channel were significantly (p<0.05)
22
!
smaller than those elicited from the wild type channel for nearly every voltage
applied in the test protocol. The peak current density of the Q1832E mutant
varied significantly from the wild type current for test voltage pulses of -50 mV, 40 mV, -30 mV, -20 mV, -10 mV, 0 mV, 10 mV, 20 mV, 30 mV, 40 mV, 50 mV
and 60 mV. Similarly, the peak current density elicited from the R1944X mutant
channel was significantly reduced when compared to the wild type for test
voltage pulses of -40 mV, -30 mV, -20 mV, -10 mV, 0 mV, 10 mV, 20 mV, 30 mV,
40 mV, 50 mV, and 60 mV. The peak current elicited from the double mutant
channel was also significantly reduced when compared to the peak current of the
wild type channel for test voltage pulses of -50 mV, -40 mV, -30 mV, -20 mV, -10
mV, 0 mV, 10 mV, 20 mV, 30 mV, 40 mV, 50 mV, and 60 mV. There was no
statistically significant difference in peak current density when comparing any two
mutated channels at any test voltage. In summary, these data demonstrate that
the Nav1.5 current density was significantly reduced by either of the mutations,
singly or when combined. Thus, the Glutamine residue at position 1832 and the
end of the C terminus are essential for proper functioning of the Nav1.5 channel.
Time Constant of Inactivation
The time constant of inactivation for the various channels was also
measured. This was done by fitting the inactivation portion of a trace current with
a double exponential equation. The function used to fit the inactivation curves
was: I = Afast(exp[&(t & k)/'fast]) + Aslow(exp[&(t & k)/'slow] + C),
23
!
where Afast and Aslow are fractions of recovery of the fast and slow
components, t is time, and k is the delay factor for activation or inactivation.
This allowed for the measurement of the fast and slow time constants of
inactivation for each channel. Table 1 shows that the majority of channels had
time constants of inactivation that were not significantly different from those of
wild-type channels. However, Nav1.5-Q1832E channels had a significantly larger
fast time constant of inactivation when compared to wild-type channels. This
could be characterized as a gain-of-function mutation, however, as the current
density produced by these channels is so drastically reduced, it is likely that the
amount of current that passes is carried in this extended time is small.
'fast
'slow
n
WT
2.6 ± 0.3
37.0 ± 11.3
8
R1944X
2.0 ± 0.1
25.1 ± 2.8
5
Q1832E
6.0 ± 1.52 *
25.2 ± 7.0
5
Double Mutant
2.1 ± 0.5
35.7 ± 7.4
5
* p<.05 compared to WT
Table 1: Time Constants of Inactivation
Time constants derived from current-voltage traces are shown. Time constant values
were acquired by analyzing traces using a double exponential fitting.
Recovery From Inactivation
In order to determine if the mutations of interest altered the gating
properties of the channels, a voltage protocol was designed to test each
channel’s recovery from inactivation. In this protocol, a potential of -20 mV was
24
!
applied to the cell in order to procure the maximal amount of current. Then, at
time points ranging from one millisecond to 500 milliseconds after the initial
activating pulse, a second test pulse of -20 mV was applied to the cell. The initial
pulse activated the channels, which subsequently inactivated. By measuring the
current density produced from each of the second test pulses, it was possible to
measure how quickly the channels recover from inactivation.
This was done by taking the ratio of the current elicited from each test
pulse to that produced from the initial activating pulse and comparing the
relationships for each. Figure 3A shows the recovery from inactivation curves for
the wild type and each of the mutant Nav1.5 channels.
When all of the relationships are viewed together there is no obvious
difference in the recovery from inactivation properties in any of the mutated
Nav1.5 channels when compared to the wild type. Indeed, neither the Nav1.5Q1832E mutant nor the double mutant differed significantly from the wild type
channel in their recovery from inactivation relationships. However, the Nav1.5R1944X channel recovered slightly, but significantly (p=0.042), slower from
inactivation compared to the wild type Nav1.5 channel.
25
!
A
C
B
1.0
Normalized Current
0.8
WT
Q1832E
ExpDec1 of WT
ExpDec1 of Q1832E
0.6
0.4
0.2
0.0
1
10
100
Time (ms)
Figure 3: Time
Courses for Recovery
from Inactivation
A) Time courses for WT,
Q1832E, R1944X and
double mutant channels
shown together. Voltage
protocol inset.
B) WT beside Q1832E
C) WT beside R1944X
D) WT beside Double
Mutant
D
26
!
Time Constant
n
WT
7.19 ± 1.25
4
Q1832E
9.20 ± 0.84
11
R1944X
11.62 ± 1.36 *
5
Double Mutant
7.12 ± 0.38
4
p<.05 compared to WT
Table 2: Recovery from Inactivation Time Constants
Time constants are shown ± SEM for recovery from inactivation time courses. Values for
time constant acquired from a single exponential fit of the data
Steady State Inactivation
To further investigate how the gating properties of Nav1.5 channels were
affected by the mutations, the steady state inactivation relationships were
measured for each of the channels being studied. This property is important
because fast inactivation of Nav1.5 channel conductance is essential for timely
membrane repolarization and a properly long refractory period. Furthermore,
even small shifts in the steady state inactivation curve can have powerful effects
on the number of Nav1.5 channels that are available to contribute to the action
potential. This is because the midpoint for the Nav1.5 current steady state
inactivation curve is relatively near the resting membrane potential, so even a
slightly shifted relationship can have severe physiological implications (Rivolta et
al., 2002).
In order to determine the steady state inactivation relationships for our
channels, the protocol in Figure 4A was designed. In this protocol, the membrane
27
!
potential was held at -180 mV before it was increased to -30 mV in a stepwise
fashion to elicit various levels of channel opening before a test pulse of -20 mV
was applied immediately afterward. By taking the ratio of current elicited from
each of the test pulses to that of the first test pulse we could determine the
steady state inactivation relationship for the channel being tested.
Again, there was no obvious difference in the steady state inactivation
relationships of the wild type and mutant channels. Figure 4B shows each of the
curves plotted alongside one another with the normalized current produced on
the y-axis and the potential used for the initial pulse on the x-axis. There is no
statistically significant difference for any of the mutant steady state inactivation
relationships when compared to the wild type.
The results from the recovery from inactivation and steady state inactivation
experiments suggest that although the mutations of interest have a profound
effect on the current that can be elicited from the channels, there is very little
difference in the gating properties of the mutant channels and the wild type
channels. As the double mutant channel did not differ significantly from the wild
type channel in its recovery from inactivation relationship nor its steady state
inactivation relationship, it can be concluded that deleterious phenotype present
in the patient did not result from a change in the gating properties of the channel.
28
!
A
B
)
Figure 4: Steady State Inactivation
A) The voltage protocol used to measure the steady state inactivation properties of each
channel.
B) Steady state voltage dependence of inactivation. Lines represent average fits of the
data with the Boltzmann function.
29
!
DISCUSSION
The data produced in this functional analysis provides an explanation of
the phenotype that may have resulted in the death of the five-week-old victim.
Through our studies we have determined that both mutations alone and the
double mutant have detrimental effects on the Nav1.5 phenotype, and this was
likely the cause of death.
Q1832E
The point mutation Q1832E had a drastic effect on the Nav1.5 channel’s
functioning. As shown by the current voltage relationship, the presence of this
single mutation alone is enough to reduce the maximum peak sodium current to
a value that is merely 17.6% of the normal value. It was expected that Q1832E
would result in a decreased current density when compared to the wild type
Nav1.5 channel, as the mutation has already been identified as one that results in
Brugada Syndrome; however, it was surprising that the Nav1.5 current was
nearly obliterated by this single mutation. Normally the Nav1.5 subunit interacts
with the fibroblast growth factor homologous family members through association
with the channel’s amino acid residues 1773 to 1832. The drastic reduction in
current may be due to a disruption in this interaction, leading to a dysfunctional
channel.
In this study we have shown that the gating properties of Nav1.5 channels
are unaffected by the Q1832E mutation, and since the current was so drastically
30
!
reduced as a result of this mutation, it seems likely that the protein is not being
expressed on the membrane at a normal level. This may be due to errors in
trafficking that result from the presence of the mutation or the channel may be
degraded at a higher level when the mutation is present. Further studies are
needed to determine mechanism that causes the current to be reduced in the
point mutated Q1832E channel.
R1944X
Similar to the Q1832E mutation, R1944X channels showed a drastically
reduced current density in the current voltage relationship. Channels expressing
this truncation mutation alone produced 26.2% of the wild type maximum peak
current density. Before starting our experimentation, we hypothesized that this
mutation would result in a greatly reduced current density, as it cuts off essential
protein interaction motifs that are present at the C terminal. Our experimentation
found a slower recovery from inactivation when the R1944X mutation was
present by itself in Nav1.5. This modification in the gating of the channel may be
due to disruption of the association of the proteins that normally interact with the
PZY domain binding motif. The drastically reduced current produced by the
truncated channel may be a result of impaired trafficking or increased protein
degradation prior to its reaching the cellular membrane. Additional experiments
are needed to test these theories.
31
!
Double Mutation
Although the analysis of Q1832E channels and R1944X channels has
provided insight into how Nav1.5 currents are affected by the individual
mutations, it is not possible to draw conclusions about the phenotype that existed
in the patient from these studies alone. In order to interpret how the patient’s
phenotype was affected by the mutations in her SCN5A gene, the results from
the experimentation on the double mutant channel must be examined.
The current voltage relationship demonstrated that there is a severe
reduction in current density in the double mutant channels when compared to the
wild type. However, this was the only tested parameter that was affected by the
double mutation, as neither the recovery from inactivation nor the steady state
inactivation properties were changed. It seems likely that the patient’s deleterious
phenotype was a result of the reduction in current and that gating changes did
not play a role in the functional alteration of the patient’s Nav1.5 channels.
Clinical Correlations
For each of the experimental channels studied, the most noticeable effect
the mutations had was a reduction in Nav1.5 current. Therefore, all three of the
mutations—Q1832E, R1944X, and the double mutant—can be described as
loss-of-function mutations. Loss-of-function mutations in Nav1.5 often result in
Brugada syndrome, and this was likely the case for our patient. Previously, it was
found that the presence of Q1832E alone could result in the disease. Our results
32
!
are in agreement with that finding, as it is feasible that the drastically reduced
current elicited from the Q1832E mutant would result in severe clinical
complications.
It is also feasible that the reduced current resulting from the double mutant
channel expressed in our patient resulted in Brugada Syndrome. As the patient
was just five weeks old and she died in her sleep, her death could be a result of
Sudden Infant Death Syndrome (SIDS). Brugada syndrome is one of the
diseases that most commonly underlies SIDS, and it seems as though this was
the case for our patient.
Limitations and Future Studies
Although this functional analysis shed light onto the underlying cause of
the patient’s death, there is still much that can be done to acquire a more
thorough understanding of the patient’s condition. In our study we discovered
that each of the mutations resulted in reduction in the function of Nav1.5, but
there are still many important questions to be answered. The reduction in current
produced in the mutant channels could be a product of a change in any of the
three following factors: the number of channels present at the membrane, the
open probability of the channels, and/ or the unitary current produced by the
channel. As single channel recordings were not carried out, it is not possible to
decisively conclude which of these factors was altered by the mutations. As the
mutations are located within protein interaction motifs, it is possible that a
33
!
disruption in these interactions is responsible for the phenotype observed.
However, it is not possible to make this conclusion based on our results, and
further experimentation should be carried out to test this hypothesis. Ideally,
immunocytochemistry assays would be used in the future to determine how
these mutated channels are being trafficked and if it differs from the trafficking of
wild type channels.
A limitation of this functional analysis is the lack of use of the (1 subunit.
The absence of this subunit prevents us from accurately measuring late
persistent current of the Nav1.5 mutants, an important factor in Long QT
Syndrome 3. Cotransfection of cells with the (1 subunit has shown to affect the
gating of Nav1.5, resulting in an earlier return to baseline after the channel has
been activated (Fahmi et al., 2001). Conclusions based on measurement of the
late persistent sodium current without the (1 subunit likely would not reflect any
meaningful physiological condition. Although it seems as though the mutations
we studied primarily produce proteins with loss-of-function effects, it would be
important to use the (1 subunit if a persistent current tests were carried out in the
future to test for gain-of-function properties.
This analysis also faced limitations due to time constraints. In steady state
inactivation experiments, the number of traces analyzed was limited to two for
experiments on the wild type channel. Although the steady state inactivation
relationship does not seem to be affected by the mutations, the analysis would
greatly benefit from more wild type recordings. However, the wild type steady
34
!
state inactivation curve procured from our limited experimentation matches very
closely to what has been measured in other experimentation (Murphy et al.,
2012).
Mutations of SCN5A and the effects they have on the Nav1.5 channel’s
function have been studied extensively. With this analysis we have aimed to
contribute to the growing wealth of knowledge on SCN5A mutations and to better
understand the disease state that was present in our patient.
35
!
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CURRICULUM VITAE
Year of Birth: 1990
Jace Morganstein
14N880 Lac du Beatrice
West Dundee, IL 60118
(847)346-4725
[email protected]
Education
University of Michigan, Ann Arbor
B.S. Anthropology. Biochemistry Minor.
August 2012
Boston University School of Medicine
M.S. Medical Sciences Candidate
May 2014
Experience
Volunteer Research Assistant, 2013-2014
Coetzee Lab at NYU Langone Medical Center, New York City, New York
Learned basic lab skills as well as some more advanced experimental
techniques. Carried out electrophysiological experiments using the patch clamp
technique and learned first hand about the importance of research in medicine.
Volunteer, 2013
Massachusetts Ear and Eye Infirmary, Boston, Massachusetts
Completed data entry projects for the employee health clinic, through which I
became proficient in Microsoft Excel. Assisted employee patients upon their
arrival to the clinic.
Student Observer, Summer 2010
Cermak Health Services, Chicago, Illinois
Shadowed a variety of general practitioners and specialists in their daily rounds
through the Hospital of the Cook County Jail. Observed interactions with inmate
patients and learned from the unique issues that frequently arise in correctional
medicine. Became knowledgeable of various diseases and how they are treated.
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Gained insight into the diverse array of professional and people skills an effective
physician must possess.
Activities
Executive Board Member, 2011-2012
College Democrats, Ann Arbor, Michigan
Demonstrated strong leadership skills as an executive of one of the largest
student organizations on campus. Formulated the agenda for the 2011-2012
school year and helped coordinate canvassing and voter registration events.
Lead and coordinated outreach events to increase voter awareness on campus.
Lobbied several state politicians and participated in numerous letter writing
campaigns and constituent phone banking events.
Co-Chair, 2011-2012
College Democrats Committee on Environmental Issues, Ann Arbor, Michigan
Led the 20+ member environmental committee of the College Democrats at the
University of Michigan. Launched a website for students to rate the efficiency of
their off campus housing and to learn ways to improve the energy efficiency of
the housing. Planned and managed a conference that included professors,
politicians and prominent community members as presenters. Participated in
numerous rallies, letter writing campaigns, voter outreach events and lobbying
efforts.
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