Download 1-Syntrophin Mutations Identified in Sudden Infant Death Syndrome

␣1-Syntrophin Mutations Identified in Sudden Infant Death
Syndrome Cause an Increase in Late Cardiac
Sodium Current
Jianding Cheng, MD, PhD; David W. Van Norstrand, BS; Argelia Medeiros-Domingo, MD, PhD;
Carmen Valdivia, MD; Bi-hua Tan, MD, PhD; Bin Ye, PhD; Stacie Kroboth, BS;
Matteo Vatta, PhD; David J. Tester, BS; Craig T. January, MD, PhD;
Jonathan C. Makielski, MD; Michael J. Ackerman, MD, PhD
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Background—Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About
5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated
mutations in ␣1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released
inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased
peak and late sodium current (INa) via S-nitrosylation of the cardiac sodium channel. This study determined the
prevalence and functional properties of SIDS-associated SNTA1 mutations.
Methods and Results—Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA
sequencing of SNTA1’s open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S,
T262P, S287R, T372M, and G460S) were identified in 8 (⬇3%) of 292 SIDS cases. These mutations were engineered
using polymerase chain reaction– based overlap extension and were coexpressed heterologously with SCN5A, neuronal
nitric oxide synthase, and PMCA4b in HEK293 cells. INa was recorded using the whole-cell method. A significant 1.4to 1.5-fold increase in peak INa and 2.3- to 2.7-fold increase in late INa compared with controls was evident for S287R-,
T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also
caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and
inactivation curves to increase window current.
Conclusions—Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the
subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel
interacting proteins such as ␣1-syntrophin from similarly rare but innocuous ones. (Circ Arrhythm Electrophysiol.
2009;2:667-676.)
Key Words: death 䡲 sudden 䡲 long-QT syndrome 䡲 genetics 䡲 ion channels 䡲 nitric oxide synthase
A
leading cause of death during the first 6 months of life
in developed countries, sudden infant death syndrome
(SIDS) is defined as the sudden death of an infant under 1
year of age that remains unexplained after a thorough case
investigation, including performance of a complete autopsy, examination of the death scene, and review of
clinical history.1 Although several hypotheses have been
formulated as potential pathogenic mechanisms for SIDS,
including apnea, airway obstruction, rebreathing of expired gases, thermal stress, infection, cardiac arrhythmia,
and neurotransmitter serotonin (5-HT)-mediated brain
stem abnormalities, SIDS remains poorly understood with
largely unknown etiology.2– 6
Clinical Perspective on p 676
More than a decade ago, an impressive clinical investigation based on 34 442 Italian newborn infants indicated that
prolongation of the QTc interval (⬎440 ms) in the first week
of life was strongly associated with SIDS.7 Soon after,
molecular evidence provided definitive evidence to link SIDS
and type 1 long-QT syndrome (LQTS).8 Recent postmortem
molecular analyses have established a pathogenic basis for
Received July 2, 2009; accepted August 19, 2009.
From the Division of Cardiovascular Medicine (J.C., C.V., B.T., B.Y., S.K., C.T.J., J.C.M.), Department of Medicine, University of Wisconsin,
Madison; the Departments of Medicine, Pediatrics, and Molecular Pharmacology and Experimental Therapeutics (D.W.V., A.M.-D., D.J.T., M.J.A.),
Mayo Clinic, Rochester, Minn; and the Section of Pediatric Cardiology (M.V.), Texas Children’s Hospital/Baylor College of Medicine, Houston, Tex.
Dr Cheng and Mr Van Norstrand contributed equally to this work.
The online-only Data Supplement is available at http://circep.ahajournals.org/cgi/content/full/CIRCEP.109.891440/DC1.
Correspondence to Michael J. Ackerman, MD, PhD, Long QT Syndrome Clinic and the Mayo Clinic Windland Smith Rice Sudden Death Genomics
Laboratory, Guggenheim 501, Mayo Clinic, Rochester, MN 55905. E-mail [email protected]
© 2009 American Heart Association, Inc.
Circ Arrhythm Electrophysiol is available at http://circep.ahajournals.org
667
DOI: 10.1161/CIRCEP.109.891440
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December 2009
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channelopathic SIDS with the identification and functional
characterization of mutations in LQTS and short-QT syndrome susceptibility genes (KCNQ1, KCNH2, SCN5A,
KCNE2, CAV3, and SCN4B) in SIDS victims9 –16 Notably,
these molecular studies suggest that LQTS mutations are
responsible for approximately 5% to 10% of SIDS, with
approximately 50% of this subset of channelopathic SIDS
stemming from mutations occurring in either the SCN5Aencoded pore-forming ␣-subunit of the Nav1.5 cardiac sodium channel (SCN5A) or channel interacting proteins
(ChIPs) of the SCN5A macromolecular complex.9,11,12
SCN5A gain-of-function mutations resulting in persistent late
sodium current (INa) provide the molecular substrate for
approximately 5% to 10% of congenital LQTS known as
LQT3,17,18 in which patients most often present with potentially lethal arrhythmias predominantly at rest or while
sleeping.
␣1-Syntrophin (SNTA1), a dystrophin-associated protein,
is the dominant syntrophin isoform in cardiac muscle. As a
scaffolding adapter with several protein interaction motifs,
SNTA1 binds to neuronal nitric oxide synthase (nNOS) and
the cardiac isoform of the plasma membrane Ca-ATPase
(PMCA4b) to form a complex in which PMCA4b inhibits
nNOS-mediated nitric oxide (NO) synthesis.19,20 Through a
PDZ domain, SNTA1 interacts with the C-terminus of
SCN5A.21 Recently, we discovered that SNTA1 connects the
pore-forming cardiac sodium channel ␣-subunit to the nNOSPMCA4b complex in cardiomyocytes and implicated SNTA1
as a novel LQTS-susceptibility gene (LQT12), whereby the
LQTS-associated mutation, A390V-SNTA1, disrupted binding with PMCA4b, released inhibition of nNOS, and accentuated both peak and late INa via S-nitrosylation of the cardiac
sodium channel.22 We also recently reported on a different
LQTS-associated mutation, A257G-SNTA1, which also demonstrated altered channel kinetics in HEK293 cells and
cardiomyocytes.23
Considering that perturbations in the Nav1.5 sodium channel complex may account for the majority of channelopathic
SIDS and our recent identification of mutations in another
sodium channel interacting protein, ␣1-syntrophin, as a novel
cause of LQTS, we hypothesized that mutations in SNTA1
may increase the risk for a malignant ventricular arrhythmia
during infancy and account for some cases of SIDS. In this
study, we aimed to determine the spectrum, prevalence, and
functional properties of SNTA1 mutations in SIDS.
Methods
Population-Based Cohort of SIDS
Two-hundred ninety-two SIDS cases derived from population-based
cohorts of unexplained infant deaths (114 girls, 177 boys, 1 unknown;
203 white, 76 black, 10 Hispanic, 2 Asian, 1 unknown; average age,
2.9⫾1.9 months; range, 6 hours to 12 months) were submitted for
postmortem genetic testing. To be rendered SIDS, the death of the
infant under age 1 year had to be sudden, unexpected, and unexplained after a comprehensive medico-legal autopsy.1 Infants whose
death was due to asphyxia or specific disease were excluded. This
study was approved by Mayo Clinic Institutional Review Board as an
anonymous study. As such, only limited medical information was
available, including sex, ethnicity, and age at death. Time of day,
medication use, and position at death were not available. By
definition, the infant’s medical history and family history were
negative.
SNTA1 Mutational Analysis
Genomic DNA was extracted from frozen necropsy tissue with the
Qiagen DNeasy Tissue Kit (Qiagen, Inc, Valencia, Calif) or from
autopsy blood with the Puregene DNA Isolation Kit (Gentra,
Minneapolis, Minn). Using polymerase chain reaction (PCR), denaturing high-performance liquid chromatography, and direct DNA
sequencing, open reading frame/splice site mutational analysis on
SNTA1 (chromosome 20q11.2, 8 exons) was performed as previously
described.24 Primer sequences, PCR conditions, and denaturing
high-performance liquid chromatography conditions are available on
request.
Plasmid Constructions of Mammalian
Expression Vectors
The cDNA of wild-type (WT) human SNTA1 gene (Genbank
accession No. NM_003098) was subcloned into pIRES2EGFP plasmid vector (Clontech Laboratories, Palo Alto, Calif). The G54R,
P56S, T262P, S287R, T372M, and G460S-SNTA1 missense mutations were incorporated into WT SNTA1 using the PCR-based
overlap-extension method as previously reported.22 The cDNAs of
nNOS (Genbank accession No. NM_052799) and PMCA4b (Genbank accession No. AY560895) were a generous gift from Solomon
H. Snyder (Johns Hopkins University) and Emanuel E. Strehler
(Mayo Clinic), respectively. All clones were sequenced to confirm
integrity and to ensure the presence of the introduced mutations and
the absence of other substitutions caused by PCR.
Chemical Reagent
The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) was
obtained from Cayman Chemical (Ann Arbor, Mich). The L-NMMA
was diluted in PBS buffer (pH 7.2) 10 minutes before use.
Mammalian Cell Transfection
The WT or mutant SNTA1 in pIRES2EGFP vector was transiently
cotransfected with expression vectors containing SCN5A (hNav1.5,
Genbank accession No. AB158469), nNOS, and PMCA4b at a ratio
of 1:4:4:4, respectively, into HEK293 cells with FuGENE6 reagent
(Roche Diagnostics, Indianapolis, Ind) according to manufacturer’s
instructions.
Electrophysiological Measurements
Macroscopic voltage-gated INa was measured 48 hours after
transfection with the standard whole-cell patch clamp method at
21°C to 23°C in the HEK293 cells. The extracellular (bath)
solution contained the following (in mM): NaCl 140, KCl 4,
CaCl2 1.8, MgCl2 0.75, and HEPES 5 and was adjusted to pH 7.4
with NaOH. The intracellular (pipette) solution contained the
following (in mM): CsF 120, CsCl2 20, EGTA 2, NaCl 5, and
HEPES 5 and was adjusted to pH 7.4 with CsOH. Microelectrodes
were manufactured from borosilicate glass using a puller (P-87,
Sutter Instrument Co, Novato, Calif) and were heat polished with
a microforge (MF-83, Narishige, Tokyo, Japan). The resistances
of microelectrodes ranged from 1.0 to 2.0 M⍀. Voltage-clamp
data were generated with pClamp software 10.2 and an Axopatch
200B amplifier (Axon Instruments, Foster City, Calif) with
series-resistance compensation. Membrane current data were
digitalized at 100 kHz, low-pass filtered at 5 kHz, and then
normalized to membrane capacitance.
Activation was measured by clamp steps of ⫺120 to 60 mV in 10
mV increments from a holding potential of ⫺140 mV. The midpoint
of activation was obtained using a Boltzmann function, where
GNa⫽[1⫹exp (V1/2⫺V)/K]⫺1, where V1/2 and k are the midpoint
and slope factor, respectively. G/GNa⫽INa (norm)/(V⫺Vrev), where
Vrev is the reversal potential and V is the membrane potential.
Steady-state inactivation was measured in response to a test depolarization to 0 mV for 24 ms from a holding potential of ⫺140 mV,
following a 1-second conditioning pulse from ⫺150 mV to 0 mV in
Cheng et al
SNTA1 Mutations in SIDS
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Table 1. Demographic Information and Functional Consequences for SNTA1 MutationPositive SIDS
Sex
Ethnicity
Nucleotide
Change
Amino
Acid Change
Protein
Location
Functional
Consequences
1
F
H
160 G⬎C
G54R
PH1
WT
4
F
B
166 C⬎T
P56S
PH1
WT
2
F
B
166 C⬎T
P56S
PH1
WT
0
F
B
166 C⬎T
P56S
PH1
WT
3
F
W
784 A⬎C
T262P
PH1
1Peak INa
Age, mo
⫹Shift inact
0.75
F
W
861 C⬎G
S287R
Linker
1Peak INa
⫹Shift inact
1Late INa
2
M
W
1115 C⬎T
T372M
PH2
1Peak INa
⫹Shift inact
1Late INa
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1
F
W
1378 G⬎A
G460S
SU
1Peak INa
⫹Shift Inact
1Late INa
F indicates female; M, male; H, Hispanic; B, black; W, white; Inact, inactivation.
10 mV increments. The voltage-dependent availability from inactivation relationship was determined by fitting the data to the
Boltzmann function: INa⫽INa-max [1⫹exp (Vc ⫺V1/2)/K]⫺1, where
V1/2 and k are the midpoint and the slope factor, respectively, and Vc
is the membrane potential. Decay rates and amplitude component
were measured from the trace beginning after peak INa at 90% of
peak INa to 24 ms and fitted with a sum of exponentials(exp): INa
(t)⫽1⫺ [Af exp(⫺t/␶f)⫹AS exp(⫺t/␶S)]⫹ offset, where t is time, and
Af and AS are fractional amplitudes of fast and slow components,
respectively.
Persistent or late INa was measured as the mean between 600 and
700 ms after the initiation of the depolarization from ⫺140 mV to
⫺20 mV for 750 ms after passive leak subtraction as previously
described.13,22,25 We have previously shown that this leak subtraction
method is comparable to saxitoxin subtraction methods.
Statistical Analysis
All data points are reported as mean and SEM. Determinations of
statistical significance were performed using a Student t test for
comparisons of 2 means or using ANOVA for comparisons of
multiple groups. Statistical significance was determined by a value
of P⬍0.05.
Results
SNTA1 Mutational Analysis in SIDS
Overall, 6 distinct, rare SNTA1 missense mutations (G54R,
P56S, T262P, S287R, T372M, and G460S) were detected in
8 of the 292 SIDS cases (2.7%; 7 girls; average age, 1.7
months; range, just after birth to 4 months; Table 1 and Figure
1A). SNTA1 mutations were identified in 7 of 114 (6.1%)
female infants compared with only 1 of 177 (0.6%) male
infants (P⬍0.01). P56S was found in 3 cases, all black
infants. Demographic data for all SNTA1 mutation-positive
SIDS is shown in Table 1. No other putative cardiac channelopathic gene mutations had been previously identified in
these 8 SIDS victims.9,13,15,16,26,27 One of the P56S-SNTA1
infants also hosted the previously described SCN5A late
INa-producing T78M-CAV3 rare polymorphism.13 In addition, 4 cases and 3 control subjects were heterozygous for the
combined variants P74L and A257G. Given the ⬇1% frequency for both cases and control subjects, P74L/A257G was
excluded from further studies due to its status as a common
polymorphism. Because of the anonymized nature of the
study, determination of genetic variants as transmitted or de
novo was not feasible.
All 6 mutations were absent in 800 reference alleles and
involved residues with various degrees of conservation across
species, with the G54R, S287R, T372M, and G460S being
most highly conserved (Figure 1B). Two of the mutations
(G54R and P56S) localized to the first pleckstrin homology 1
(PH1) domain (amino acids 1 to 80), 1 mutation (T262P)
localized to the second PH1 domain (aa 161 to 263), 2 mutations
(S287R and T372M) localized either in or very near the
pleckstrin homology 2 (PH2) domain (aa 292 to 399), and 1
mutation (G460S) localized to the syntrophin unique (SU)
domain (aa 447 to 503) of SNTA1 (Figure 1C and 1D).
SNTA1 Mutations in SIDS Increase Peak and Late
INa in HEK293 Cells
Functional characterization of SNTA1 mutations was performed
in HEK293 cells, which transiently expressed SCN5A, nNOS,
PMCA4b, and either the wild-type SNTA1 (WT-SNTA1) or the
mutant SNTA1. Compared with WT-SNTA1, 4 of the 6 SNTA1encoded missense mutations: T262P-, S287R-, T372M-, and
G460S-SNTA1, had significantly larger peak INa amplitudes,
whereas G54R- and P56S-SNTA1 were similar to WT-SNTA1
(Figure 2A and 2B and Table 2).
We measured the level of persistent/late INa as a percentage
of peak INa elicited by prolonged depolarization and leak
subtraction. Compared with WT-SNTA1, S287R-, T372M-,
and G460S-SNTA1 caused a significant 2.3- to 2.7-fold
increase in late INa, whereas the other 3 missense mutations
(G54R-, P56S-, and T262P-SNTA1) were comparable to
WT-SNTA1 (Figure 2C and 2D and Table 2).
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Figure 1. Identification and location of
SNTA1 mutations in SIDS. A, DNA
sequence chromatograms; B, sequence
homologies for SIDS-associated SNTA1
mutations; C, linear topology for SNTA1
with mutation localization; D, diagram
shows SCN5A-SNTA1-nNOS-PMCA4b
complex with location of interaction
between SNTA1 and complex subunits.
*Previously reported LQT12-associated
mutations.
The Sodium Channel Gain of Function Caused by
the 3 SIDS-Associated SNTA1 Mutations Is
nNOS-SNTA1-PMCA4b Complex Dependent
To observe the effect of NOS inhibitor on the PMCA4bnNOS-SNTA1-SCN5A complex, L-NMMA (100 ␮mol/L)
was introduced into the HEK293 cell culture medium 12
hours before testing. The marked accentuation in late INa
precipitated by S287R-, T372M-, and G460S-SNTA1 was
abolished by L-NMMA, and the corresponding peak INa were
also reversed (Table 2). These results indicated that akin to
the original LQT12-associated mutation, A390V, NO was the
key factor by which SNTA1 affected function. To determine
whether these SNTA1 mutations cause abnormal INa through
modulation of its interaction with SCN5A, we performed
functional characterization of SIDS-associated SNTA1 mu-
tations using HEK293 cells coexpressing only SCN5A and
either the WT-SNTA1 or the SNTA1 mutants. None of the 6
SNTA1 mutations coexpressed with SCN5A alone showed a
significant difference in peak INa, late INa, or channel kinetics
compared with WT-SNTA1 (supplemental Table 1).
To clarify whether PMCA4b (ie, the full nNOSPMCA4b-SNTA1 complex) is required for SNTA1
mutation-mediated effects on SCN5A function, we tested
HEK293 cells only coexpressing SCN5A, nNOS, and
either the WT-SNTA1 or the SNTA1 mutants, without
PMCA4b expression. Again, none of the mutations showed
a significant difference in peak INa, late INa, or channel
kinetics compared with WT-SNTA1 (supplemental Table
2). These data suggest that the sodium channel gain of
function caused by the 3 SIDS-associated SNTA1 mutants
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SNTA1 Mutations in SIDS
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Figure 2. Electrophysiological properties of cardiac sodium channel in HEK293 cells coexpressing PMCA4b, nNOS, and either WT or
mutant SNTA1. A, Representative whole-cell current traces showing increased peak INa associated with G460S-, T372M-, S287R-, and
T262P-SNTA1; B, summary data of peak INa densities from every group; C, representative traces showing increased late INa associated
with G460S-, T372M-, and S287R-SNTA1 compared with WT-SNTA1; D, summary data of late INa normalized to peak INa after leak
subtraction. The number of tested cells is indicated above the bar. *P⬍0.05 versus WT-SNTA1.
with the WT-SNTA1. Although none of the SNTA1 mutations showed a significant difference in activation parameters
compared with WT-SNTA1 (Figure 3A and Table 2), the
T262P-, S287R-, T372M-, and G460S-SNTA1 mutations
caused a statistically significant depolarizing shift in inactivation (Figure 3B and Table 2). For the mutants S287R-,
T372M-, and G460S-SNTA1, the increase in overlap of the
activation and inactivation curves resulted in the increase of
is mediated by the entire nNOS-PMCA4b-SNTA1
complex.
SNTA1 Mutations Changed Sodium Channel
Gating Properties Through an
nNOS-Dependent Mechanism
We analyzed the kinetic parameters of activation and inactivation of all 6 SNTA1 mutations and compared these data
Table 2. Electrophysiological Properties of Sodium Channels in HEK293 Cells Coexpressing SCN5A, PMCA4b, nNOS, and Either WT or
Mutant SNTA1
Peak INa
Samples
Activation
Inactivation
Late INa
pA/pF
N
V1/2, mV
K
n
V1/2, mV
K
n
%
n
WT-SNTA1
⫺185⫾18
33
⫺40.7⫾0.9
4.0
30
⫺77.4⫾0.9
5.0
30
0.155⫾0.03
30
G460S-SNTA1
⫺254⫾23*
12
⫺39.8⫾1.1
4.0
12
⫺72.8⫾0.8*
5.5
12
0.361⫾0.06*
12
13
T372M-SNTA1
⫺271⫾28*
13
⫺40.8⫾1.0
4.1
12
⫺74.9⫾0.5*
5.1
13
0.412⫾0.07*
S287R-SNTA1
⫺284⫾35*
14
⫺42.5⫾2.3
4.2
9
⫺74.0⫾1.4*
5.0
12
0.357⫾0.05*
12
T262P-SNTA1
⫺335⫾51*
17
⫺40.3⫾0.6
4.1
9
⫺74.1⫾0.7*
5.0
16
0.223⫾0.04
12
P56S-SNTA1
⫺221⫾30
14
⫺42.8⫾1.4
4.3
14
⫺79.4⫾0.9
5.0
14
0.080⫾0.04
14
10
G54R-SNTA1
⫺240⫾40
12
⫺39.9⫾1.0
4.0
9
⫺76.0⫾1.0
5.0
9
0.148⫾0.04
G460S-SNTA1⫹L-NMMA
⫺231⫾39
6
⫺43.0⫾1.7
4.2
5
⫺78.9⫾1.2
5.3
6
0.167⫾0.07
5
T372M-SNTA1⫹L-NMMA
⫺228⫾34
8
⫺41.7⫾2.1
4.3
5
⫺76.7⫾1.1
5.0
8
0.203⫾0.04
7
S287R-SNTA1⫹L-NMMA
⫺258⫾41
6
⫺41.4⫾1.4
4.0
5
⫺77.0⫾1.6
5.1
6
0.194⫾0.06
5
*P⬍0.05 vs WT-SNTA1.
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Figure 3. Voltage-dependent gating for SCN5A coexpressed with PMCA4b, nNOS, and either WT or mutant SNTA1. A, None of the 6
SNTA1 mutations altered steady-state activation parameters significantly. B, G460S-, T372M-, S287R-, and T262P-SNTA1 caused a
statistically significant depolarizing shift in inactivation by 2.5 to 4.6 mV. The peak current activation data are replotted as a conductance (G) curve with steady-state inactivation relationships to show C, G460S-; D, T372M-; and E, S287R-SNTA1 increase the overlap
of these relationships. Lines represent fits to Boltzmann equations with parameters of the fit and n numbers in Table 2. Triangles represent inactivation curves for WT (filled) and mutant (open), whereas the filled boxes and open circles represent activation curves for WT
and mutant, respectively. The window area of each mutant (right-slanted line area under curves) was significantly enhanced beyond WT
(left-slanted line area under curves).
the “window current” (Figure 3C, 3D, and 3E). Time constants (␶f, ␶s) were obtained from 2-exponential fits of decay
phase of macroscopic INa measured at various test potentials.
Compared with WT-SNTA1, the S287R-, T372M-, and
G460S-SNTA1 mutations showed significantly larger ␶f val-
ues across a wide range of test potentials (Figure 4), indicating that fast inactivation was impaired and sodium current
decay was slower. There was no difference in time constant ␶s
or fractional amplitudes for the 2 time constants observed.
Notably, the inactivation parameters (Table 2) and time
Figure 4. Decay of macroscopic current and voltage dependence of inactivation fast time constants. A, Representative normalized
whole-cell current traces at ⫺20 mV showing slower decay in G460S-, T372M, and S287R-SNTA1 compared with WT-SNTA1. B, Compared with WT-SNTA1, G460S-, T372M-, and S287R-SNTA1 showed significantly larger fast component (␶f) values across a wide range
of test potentials from ⫺20 mV to 10 mV except T372M, where deviations from WT were from ⫺10 to 10 mV. *P⬍0.05 versus
WT-SNTA1.
Cheng et al
constants ␶f of S287R-, T372M-, and G460S-SNTA1 (data
not shown) returned to normal levels after the application of
L-NMMA, suggesting the alteration of channel gating properties caused by these mutants was mediated by an NOdependent mechanism akin to the NO-dependent effect of
these 3 particular SNTA1 missense mutations on both peak
and persistent sodium current. Last, there were no significant
differences between WT and mutants in recovery from
inactivation (data not shown).
Discussion
SNTA1: A Novel Susceptibility Gene for SIDS
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Cardiac channelopathies, especially LQTS, have been shown
to account for up to 10% of SIDS.8,9 So far, mutations in 8
cardiac channelopathy-susceptibility genes have been implicated in the pathogenesis of SIDS. Four of these genes encode
cardiac ion channel ␣-subunits (SCN5A, KCNQ1, KCNH2,
and RYR2), 3 encode ion channel ␤-subunits (KCNE2,
SCN3B, and SCN4B), and 2 encode other channel-interacting
proteins (CAV3, GPD1L).9,11–16,26,27 Most recently, the
SNTA1-encoded sodium ChIP, ␣1-syntrophin, a key component of the PMCA4b-NOS-SNTA1-SCN5A macromolecular
complex, was implicated as a new LQTS-susceptibility gene
by our study group.22,23
In the present study, we provide molecular and functional
evidence implicating SNTA1 as a novel susceptibility gene for
SIDS. In total, 6 SNTA1 missense mutations (G54R, P56S,
T262P, S287R, T372M, and G460S) were identified in 8
SIDS cases, with 1 particular mutation, P56S, identified in 3
unrelated cases. Interestingly, 7 of the 8 mutations were
found in girls. Overall, nearly 3% of SIDS victims hosted
these putative SNTA1 mutations that were absent in more than
800 reference alleles, localized to presumably key functional
domains of ␣1-syntrophin, and mostly involved highly conserved residues across a variety of species (P56S and T262P
were less conserved). Although all 6 variants met the pathogenic criteria of being “rare” and localized to key functional
domains, only 3 of the missense mutations (S287R, T372M,
and G460S) significantly perturbed the cardiac sodium channel. Electrophysiological studies showed the S287R-,
T372M-, and G460S-SNTA1 mutations resulted in a significant increase in both peak and late INa in HEK293 cells
through the PMCA4b-NOS-SNTA1-SCN5A macromolecular
complex. The remarkable gain of function of the sodium
channel caused by these 3 SNTA1 mutants were similar to
that observed in other SCN5A mutation-positive LQT3 patients and the A390V-SNTA1 mutation positive LQTS patient previously described.22 The other 3 variants (G54R,
P56S, and T262P), although considered functionally insignificant in the modification of SCN5A channel biology and now
classified as a functional insignificant variant, may have
helped nevertheless to elucidate which functional domains of
SNTA1 are most important in maintaining integrity and
proper function of the SCN5A-nNOS-SNTA1-PMCA4b
complex. Last, of the 3 functionally significant variants, 2
were found in girls and 1 in boys, minimizing any potential
sex effect on risk of sudden death in SNTA1 mutation-positive
individuals.
SNTA1 Mutations in SIDS
The Disturbance in nNOS-SNTA1-PMCA4b
Complex Relieved the Inhibition of nNOS
by PMCA4b
673
Like other syntrophin isoforms (␤1, ␤2, ␥1, and ␥2), SNTA1
(␣1) comprises 4 conserved domains, 2 pleckstrin homology
domains (PH1 and PH2) that are involved in the recruitment
of proteins to the sarcolemma,28 a PDZ domain that inserts
within PH1 and has been shown to bind to nNOS and
SCN5A,21,29 and a syntrophin unique COOH-terminal domain (SU) that binds SNTA1 to dystrophin.30 The fact that
there are up to 4 SNTA1 binding sites in close proximity
within a single dystrophin complex31 suggests that SNTA1
probably brings several signaling molecules together to form
a large signaling complex.20
In cardiomyocytes, the activity of nNOS was confirmed to
be negatively regulated by PMCA4b through direct interaction mediated by a PDZ domain.19 When SNTA1 was
introduced to the nNOS-PMCA4b complex to form the
bigger complex nNOS-SNTA1-PMCA4b, the maximal inhibitory effect of PMCA4b on nNOS was observed compared
with the nNOS-PMCA4b complex, suggesting that the interaction of SNTA1 and PMCA4b, as well as the formation of
the entire complex were critical for PMCA4b-mediated inhibition of nNOS.20
Previously, we showed the existence of the macromolecular complex SCN5A-nNOS-SNTA1-PMCA4b (Figure 1C
and 1D) in cardiomyocytes and found that the LQTSassociated A390V-SNTA1 mutation disrupted binding with
PMCA4b, released inhibition of nNOS, and consequently
increased the peak and late INa via S-nitrosylation of the
cardiac sodium channel mediated by local increased NO
concentration.22
In this investigation, 3 of the 6 SNTA1 missense mutations
(S287R, T372M, and G460S) demonstrated similarly pronounced gain-of-function effects on NaV1.5 through the
nNOS-SNTA1-PMCA4b macromolecular complex. Interestingly, some structure-function observations emerge when
comparing the domain localization of the 3 missense mutations with a distinct pathological phenotype to the 3 missense
mutations that were essentially indistinguishable from WTSNTA1. The functionally significant S287R, T372M, and
G460S-SNTA mutations were located in or very close to the
region between PH2 and SU domains, which was identified as
the region of interaction for SNTA1 and PMCA4b. The T262P
mutant with only increased peak INa was near to the binding
area, whereas the 2 WT-like mutations (P56S and G54R)
localized outside of the specific binding area (Figure 1C).
The fact that the nNOS inhibitor L-NMMA eliminated the
increased late INa caused by the S287R-, T372M-, and G460SSNTA1 mutations further supports the idea that these mutations
increase late INa in an nNOS-dependent manner. Moreover, the
functional studies for the complex SCN5A-SNTA1 (lacking
both nNOS and PMCA4b) or SCN5A-SNTA1-nNOS complex
(lacking PMCA4b) suggest that the 3 SNTA1 mutations do
not cause increased late INa by a direct interaction between
SNTA1 and SCN5A or between SNTA1 and nNOS. Based on
these data, we speculate that the 3 mutations may disturb the
interaction of PMCA4b and SNTA1 in the whole macromolecular complex SCN5A-nNOS-SNTA1-PMCA4b, thus re-
674
Circ Arrhythm Electrophysiol
December 2009
lieving the negative regulation of PMCA4b on nNOS and
thereby resulting in an increase of local NO concentrations
and a biophysical modification of the sodium channel.
Molecular Mechanism for Increased Late INa
Associated With NO Modulation
Downloaded from http://circep.ahajournals.org/ by guest on August 3, 2017
There is still some disagreement regarding the reported
modulatory effect of NO on the sodium channel, in part due
to tissue specificity and NO delivery method.32–36 Relatively
high concentrations of exogenous NO reduce peak INa in
cardiomyocytes via a cGMP associated pathway and have no
effect on activation, inactivation, or reactivation kinetics.32 In
a different study, persistent INa in rat hippocampal neurons
increased by 60% to 80% through a direct action of NO on the
sodium channel protein or on a closely associated regulatory
protein in the plasma membrane (S-nitrosylation pathway).33
Still another study in nerve terminals and ventricular myocytes showed that NO reduced the inactivation of the sodium
channel, increasing persistent INa. Further investigation confirmed the effect was independent of the cGMP pathway and
was blocked by N-ethylmaleimide, suggesting the
S-nitrosylation pathway. Importantly, in myocytes, persistent
INa was also enhanced by endogenous NO generated enzymatically by NOS, whereas NOS inhibitors abolished the
increase of both NO and persistent INa.34
The present study showed that in the presence of an nNOS
inhibitor, the marked accentuation in late INa caused by
S287R-, T372M, and G460S-SNTA1 decreased to WTSNTA1 levels and that the increase in peak INa was also
reversed. Moreover, the mutant properties of the sodium
channel (ie, positive shift of inactivation and slowing of
current decay) that underlie increased late INa were also
reversed by an nNOS inhibitor. These findings were similar
to other studies33,34 and strongly support the contention
that endogenous NO generated enzymatically by NOS is
the key signaling molecule by which SNTA1 mutants
increase peak and late INa. Our group previously showed
that A390V-SNTA1 released the inhibition of nNOS, thus
increasing endogenous NO, which in turn caused increased
direct S-nitrosylation of SCN5A compared with WTSNTA1.22 With these data, we demonstrate that the direct
S-nitrosylation effect of the increased endogenous NO
caused by SNTA1 mutations associated with SIDS can
change the characteristics of the cardiac sodium channel
and modulate late INa under physiological and pathophysiological conditions.
nNOS plays an important role in modulating the late INa
underlying sodium channel-mediated LQTS and sudden unexplained cardiac death. Notably, the previously reported
influences of common variation involving the neuronal nitric
oxide synthase adaptor protein (NOS1AP, an nNOS regulator) on QT interval duration37– 41 and most recently the
observed association of NOS1AP genetic variants with sudden cardiac death42 as well as SIDS43 have confirmed the
important role of nNOS in LQTS-related disorders. Thus, the
deeper association of nNOS complex–related proteins (for
example nNOS regulators like PMCA4b) as potential candidate genes for LQTS and sudden cardiac death deserves
further study.
Study Limitations
Implications of nNOS Complex in Sudden
Cardiac Death
Although we established a distinct association between SIDS
and SNTA1 mutations by molecular and functional evidence,
there are some limitations in the present study. First, because
these mutations were detected in a “retrospective”
population-based postmortem cohort, it is not possible to
infer true causality but only demonstrate the association of a
“proarrhythmic” genotype with certain SIDS victims. Obviously, by the nature of the study design, there are no
implantable loop recordings showing an exit rhythm of
ventricular fibrillation in the 3 infants who hosted one of
these 3 rare and functionally significant SNTA1 missense
mutations.
Second, the electrophysiological data were generated by in
vitro experiments using HEK293 cells coexpressing the
macromolecular complex SCN5A-SNTA1-nNOS-PMCA4b,
which is somewhat different from the physiological environment in human cardiomyocytes. Because ␣1-syntrophin is a
scaffolding adapter with several protein interaction motifs, it
may interact with other signaling molecules involved with
SCN5A or other ion channel complexes and therefore we
cannot exclude that these particular SIDS-associated SNTA1
mutations might exert other effects in a more native cardiomyocyte environment. However, given the demonstration of
increased late INa with the original LQTS-associated A390VSNTA1 in cardiomyocytes,22 we expect that results for these
mutations in a more native environment would demonstrate
similar findings.
Last, 3 of the 8 SNTA1-positive SIDS cases also had the
common SCN5A polymorphism, H558R, which has been
shown to alter the disease phenotype for various SCN5A
disease-associated mutations.44 – 47 Whether or not common
channel polymorphisms affect the nitrosylation pathway represents a possible future direction for this work.
The present study demonstrates a new arrhythmic cause for
approximately 1% of SIDS, characterized by increased late
INa originating from the disturbance of the nNOS complex,
and further establishes perturbations throughout the Nav1.5
sodium channel complex as the final common pathway for
the majority of channelopathic SIDS. The functional data
involving 3 of the SIDS-associated SNTA1 mutations
(S287R, T372M, and G460S) has provided additional evidence for implicating SNTA1 as a LQTS-susceptibility gene.
Most importantly, these findings strongly suggested that
In conclusion, this study implicates SNTA1 as a novel
SIDS-susceptibility gene, whereby mutant SNTA1 disturbs
the nNOS-SNTA1-PMCA4b-SCN5A complex, releasing inhibition of associated nNOS by PMCA4b and resulting in
increased peak and late INa via the upregulated endogenous
NO. This current study adds to the growing body of literature
implicating channelopathies as causing up to 10% of SIDS,
with a significant portion of channelopathic SIDS stemming
Conclusion
Cheng et al
from perturbations in the Nav1.5 cardiac sodium channel
macromolecular complex.
Sources of Funding
This work was supported by the Mayo Clinic Windland Smith Rice
Comprehensive Sudden Cardiac Death Program (M.J.A.), the University of Wisconsin Cellular and Molecular Arrhythmia Research
Program (J.C.M.), and by grants HD42569 (M.J.A.), HL60723
(C.T.J.), and HL71092 (J.C.M.) from the National Institutes of
Health.
Disclosures
None.
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CLINICAL PERSPECTIVE
Every year, more than 2000 infants in the United States die of sudden infant death syndrome (SIDS), a multifactorial event
with environmental and genetic factors converging on the vulnerable infant during the first year of life. The QT hypothesis,
first proposed in 1976, attributes a significant number of cases of SIDS to congenital cardiac channelopathies, such as
long-QT syndrome (LQTS). Approximately 5% to 10% of SIDS may be precipitated by mutations in genes encoding
proteins comprising the sodium channel macromolecular complex, including the sodium channel ␣-subunit, caveolin-3, and
GPD1L. This report implicates the recently discovered LQTS-susceptibility gene SNTA1, which encodes the structural
protein ␣1-syntrophin, as a novel potential cause of channelopathic SIDS. In vitro functional studies demonstrated that 3
of the 6 rare SNTA1 variants markedly accentuated the late sodium current consistent with an LQT3-like proarrhythmic
substrate. Interestingly, this cellular phenotype is neuronal nitric oxide synthase dependent and reversible with a neuronal
nitric oxide synthase inhibitor. Moreover, mutational effects were protein region– dependent, with the functionally
significant, SIDS-associated mutations localizing near the syntrophin binding domain with PMCA4b, an interaction
required to exert PMCA4b inhibition of neuronal nitric oxide synthase. The significance of these findings is 2-fold. First,
this work contributes to the growing body of literature implicating the cardiac channelopathies and the sodium channel
macromolecular complex as the pathogenic substrate for a small subset of SIDS victims, that is, channelopathic SIDS.
Second, given the increasing awareness of sequence variation in the general population, this work highlights the importance
of concomitant functional studies to further discern rare “deleterious” genetic variants from similarly rare yet “innocuous”
ones.
α1-Syntrophin Mutations Identified in Sudden Infant Death Syndrome Cause an Increase
in Late Cardiac Sodium Current
Jianding Cheng, David W. Van Norstrand, Argelia Medeiros-Domingo, Carmen Valdivia,
Bi-hua Tan, Bin Ye, Stacie Kroboth, Matteo Vatta, David J. Tester, Craig T. January, Jonathan
C. Makielski and Michael J. Ackerman
Downloaded from http://circep.ahajournals.org/ by guest on August 3, 2017
Circ Arrhythm Electrophysiol. 2009;2:667-676; originally published online September 12, 2009;
doi: 10.1161/CIRCEP.109.891440
Circulation: Arrhythmia and Electrophysiology is published by the American Heart Association, 7272 Greenville
Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 1941-3149. Online ISSN: 1941-3084
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circep.ahajournals.org/content/2/6/667
Data Supplement (unedited) at:
http://circep.ahajournals.org/content/suppl/2009/09/12/CIRCEP.109.891440.DC1
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SUPPLEMENTAL MATERIAL
Supplemental Table 1. Electrophysiological properties of sodium channels in HEK293 cells co-expressing SCN5A and either WT or
mutant SNTA1
Peak I Na†
pA/pF
-335 ± 35
n
6
Activation
V1/2(mV)
K
-43.4 ± 1.8 4.0
n
5
Inactivation
V1/2(mV)
K
-75.4 ± 0.9
5.0
G460S-SNTA1
-294 ± 73
6
-45.1 ± 2.0
4.0
5
-77.9 ± 0.5
T372M-SNTA1
-315 ± 76
6
-42.8 ± 1.4
4.3
5
S287R-SNTA1
-348 ± 60
6
-44.6 ± 1.5
4.1
T262P-SNTA1
-254 ± 78
6
-41.4 ± 2.2
P56S-SNTA1
-307 ± 34
6
G54R-SNTA1
-326 ± 46
6
Samples
WT-SNTA1
† I Na = Sodium current
n
5
Late I Na
%
0.216 ± 0.04
n
5
5.2
5
0.245 ± 0.04
6
-75.9 ± 0.8
5.0
6
0.251 ± 0.05
6
5
-77.3 ± 1.3
5.1
6
0.194 ± 0.04
6
4.2
5
-74.7 ± 0.9
5.0
6
0.238 ± 0.05
5
-43.7 ± 1.8
4.0
5
-76.5 ± 0.7
5.3
5
0.182 ± 0.07
6
-44.0 ± 1.1
4.0
5
-74.2 ± 1.2
5.0
5
0.183 ± 0.04
6
Supplemental Table 2. Electrophysiological properties of sodium channels in HEK293 cells co-expressing SCN5A, nNOS and either
WT or mutant SNTA1
Peak I Na†
pA/pF
-311 ± 60
n
5
Activation
V1/2(mV)
K
-43.7 ± 2.0 4.0
n
5
Inactivation
V1/2(mV)
K
-76.8 ± 1.4
5.5
G460S-SNTA1
-345 ± 52
5
-42.4 ± 1.6
4.2
5
-77.6 ± 1.2
T372M-SNTA1
-366 ± 63
5
-43.5 ± 1.7
4.0
5
S287R-SNTA1
-302 ± 46
5
-44.9 ± 2.1
4.0
T262P-SNTA1
-291 ± 44
5
-45.2 ± 1.0
P56S-SNTA1
-338 ± 33
6
G54R-SNTA1
-323 ± 48
5
Samples
WT-SNTA1
† I Na = Sodium current
n
5
Late I Na
%
0.473 ± 0.13
n
5
5.0
5
0.426 ± 0.12
5
-78.2 ± 1.0
5.0
5
0.395 ± 0.14
5
5
-78.5 ± 1.5
5.0
5
0.458 ± 0.16
5
4.0
5
-78.9 ± 1.4
5.1
5
0.373 ± 0.17
5
-41.6 ± 1.0
4.0
5
-75.2 ± 1.1
5.0
5
0.504 ± 0.11
6
-44.3 ± 1.4
4.1
5
-76.4 ± 0.9
5.0
5
0.481 ± 0.15
5