© 2002 Oxford University PressHuman Molecular Genetics, 2002, Vol. 11, No. 3
Genetic and biophysical basis of sudden unexplained
nocturnal death syndrome (SUNDS), a disease allelic to
Matteo Vatta1, Robert Dumaine4, George Varghese1, Todd A. Richard4, Wataru Shimizu5,
Naohiko Aihara5, Koonlawee Nademanee6, Ramon Brugada2, Josep Brugada7,
Gumpanart Veerakul9, Hua Li1, Neil E. Bowles1, Pedro Brugada8, Charles Antzelevitch4 and
Jeffrey A. Towbin1,3,*
1Department of Pediatrics (Cardiology), 2Department of Medicine (Cardiology) and 3Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX, USA, 4Masonic Medical Research Laboratory, Utica, NY,
USA, 5Department of Internal Medicine (Cardiology), National Cardiovascular Center, Osaka, Japan, 6Department of
Medicine (Cardiology), University of Southern California, Los Angeles, CA, USA, 7Cardiovascular Institute, Hospital
Clinic, University of Barcelona, Spain, 8Cardiovascular Center, OLV Hospital, Aalst, Belgium and 9Department of
Cardiology, Bhumibol Adulyadej Hospital, RTAF, Bangkok, Thailand
Received November 20, 2001; Accepted November 28, 2001
Sudden unexplained nocturnal death syndrome (SUNDS), a disorder found in southeast Asia, is characterized
by an abnormal electrocardiogram with ST-segment elevation in leads V1–V3 and sudden death due to ventricular
fibrillation, identical to that seen in Brugada syndrome. We screened patients with SUNDS for mutations in
SCN5A, the gene known to cause Brugada syndrome, as well as genes encoding ion channels associated with the
long-QT syndrome. Ten families were enrolled, and screened for mutations using single-strand DNA conformation
polymorphism analysis, denaturing high-performance liquid chromatography and DNA sequencing. Mutations
were identified in SCN5A in three families. One mutation, R367H, lies in the first P segment of the pore-lining
region between the DIS5 and DIS6 transmembrane segments of SCN5A. A second mutation, A735V, lies in the
first transmembrane segment of domain II (DIIS1) close to the first extracellular loop between DIIS1 and DIIS2,
whereas the third mutation, R1192Q, lies in domain III. Analysis of these mutations in Xenopus oocytes
showed that the R367H mutant channel did not express any current and the likely effect of this mutation is to
depress peak current due to the loss of one functional allele. The A735V mutant expressed currents with steady
state activation voltage shifted to more positive potentials. The R1192Q mutation accelerated the inactivation of the
sodium channel current. Both mutations resulted in reduced sodium channel current (INa) at a time corresponding to
the end of phase 1 of the action potential, as described previously in the Brugada syndrome. Based upon these
observations we suggest that SUNDS and Brugada syndrome are phenotypically, genetically and functionally
the same disorder.
Sudden unexplained nocturnal death syndrome (SUNDS) is a
disorder found in southeast Asia, particularly Thailand, Japan,
Philippines and Cambodia, which causes sudden cardiac death
(usually in males) during sleep (1–3). This disorder, which is
the most common cause of ‘natural’ death in the young,
healthy Asian population, is called by many descriptive terms
in the various countries, including pokkuri (‘sudden
unexplained death at night’) in Japan, lai-tai (‘died during
sleep’) in Thailand, and bangungut (‘moaning and dying
during sleep’) in the Philippines.
The clinical features of SUNDS include ST-segment
elevation in the right precordial leads (V1–V3), inconsistently
associated with right bundle branch block (RBBB) (Fig. 1)
(2,3) and ventricular tachycardia and fibrillation (VF) on
surface electrocardiogram (ECG). These clinical characteristics
are similar to those of the Brugada syndrome, a disorder
diagnosed in individuals of European descent (4–6).
The genetic cause of the Brugada syndrome was initially
described by our group and shown to be due to mutations in the
cardiac sodium channel gene, SCN5A. This has now been
confirmed by others (7,8). In previous publications, we and
others demonstrated that mutant sodium channels have a
reduced sodium channel current (INa) due to rapid inactivation
of the current or failure of the channel to express currents
(9,10). Due to the apparent clinical similarities between
*To whom correspondence should be addressed at: Pediatric Cardiology, Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030, USA.
Tel: +1 713 798 7342; Fax: +1 713 798 8085; Email: email@example.com
338 Human Molecular Genetics, 2002, Vol. 11, No. 3
Brugada syndrome and SUNDS, we speculated that these
could be allelic disorders (or even the same disease). In this
report, we describe the molecular analysis of patients with
SUNDS and identify mutations in SCN5A, confirming this
disorder to be genotypically, phenotypically and functionally
identical to Brugada syndrome.
Ten probands with clinical evidence of SUNDS were screened
for mutations in KVLQT1 (11), HERG (11), SCN5A (9,11),
minK (12) and MiRP1 (13) using single-strand DNA conformation
polymorphism (SSCP) analysis, denaturing high-performance
liquid chromatography (DHPLC) and DNA sequencing. In
three patients (M030, M032, M033), SCN5A mutations were
identified. No mutations were identified in any of the other
genes screened. Patient M030 is a sporadic case, whereas the
other two cases were probands of families. One of these was a
family with multiple affected individuals (family M032), and
apparent autosomal dominant inheritance. The second family
(M033) included a pair of affected Japanese dizygotic twins.
One twin died unexpectedly during sleep at 4 months of age.
The other twin had frequent VF episodes at 6 months of age
and has been discussed previously (14). This living child and
other family members were studied.
Patient M030. An abnormal conformer was identified in exon 9
of SCN5A in this sporadic case (Fig. 2A) and confirmed by
DHPLC analysis (Fig. 2C). DNA sequence analysis revealed a
G→A base substitution (G1100A) in exon 9 (Fig. 2B) leading
to the substitution of an arginine by a histidine at codon 367
(R367H), which lies in the first P segment belonging to the
Figure 1. ECG of SUNDS. Note the ST-segment elevation in leads V1–V3 in the proband of family M032, identical to the findings in Brugada syndrome.
Figure 2. Mutation detection in sporadic case M030. (A) SSCP analysis
demonstrates an abnormal conformer in the affected individual. (B) DNA
sequence analysis identifies a G→A substitution in exon 9 of SCN5A, resulting
in an amino acid substitution of arginine to histidine at codon 367 (R367H).
(C) DHPLC confirms an abnormal migration pattern in the affected individual.
Human Molecular Genetics, 2002, Vol. 11, No. 3
pore-lining region between the DIS5 and DIS6 transmembrane
segments of the cardiac sodium channel (Fig. 3). The P segment is
a conserved region within the Na+-channel family (15). This
mutation was not found in other family members (including
the parents) nor in 100 control individuals (200 chromosomes)
of Asian descent (data not shown).
Family M032. A point mutation was identified in the proband
of family M032. SSCP (Fig. 4A) and DHPLC analysis
(Fig. 4C) identified an abnormality in exon 14, due to a C→T
substitution (C2204T) (Fig. 4B) leading to a substitution of
alanine by valine at codon 735 (A735V). This mutation lies in
the first transmembrane segment of domain II (DIIS1) at the
border with the first extracellular loop between DIIS1 and
DIIS2 (Fig. 3). Screening of other family members identified
the same mutation in the father and the son of the proband,
both of whom had ECG abnormalities consistent with SUNDS.
The unaffected family members and 100 control patients did
not carry this mutation.
Family M033. A point mutation was identified in the proband
of family M033, the surviving dizygotic twin. DHPLC analysis
(Fig. 5A) identified an abnormality in exon 20, due to a G→A
substitution (G3575A) (Fig. 5B) leading to a substitution of
arginine by glutamine at codon 1192 (R1192Q). This mutation
lies in the intracellular loop connecting the DIIS6 to DIIIS1
(Fig. 3). Screening of other family members identified the
same mutation in the affected family members (data not
shown). The unaffected family members and 100 control
patients did not carry this mutation.
To study the mechanism by which these mutations may
contribute to the syndrome, heterologous expression in
Xenopus oocytes was performed. The mutant channel R367H
did not express any current and the likely effect of this
mutation is to depress INa due to the loss of one functional
allele (Fig. 6A and C).
In contrast, expression of the A735V mutant appeared
normal (Fig. 6B). Although visual inspection of the current
traces suggested faster decay of A735V current, statistical
analysis of the current decay fit to the sum of two exponential
failed to show a significant increase of inactivation kinetics
(Z-test for mean or ANOVA). However, the time to peak of
the maximum current was significantly briefer for A735V
(1.8 ± 0.1 ms) versus wild-type (WT) (2.2 ± 0.1 ms) (Fig. 7).
The average maximum current was measured at –10 and –5 mV
for WT and A735V, respectively. The shorter time to peak and
shift of the voltage of maximum current for A735V was due to
a 7 mV shift of the steady state activation curve towards more
positive potentials (Fig. 7B).
Figure 8 shows that the shift in steady state activation leads
to a significant reduction in A735V current at a time (3 ms) and
over a range of potentials (–30 to 0 mV) corresponding to
phase 1 of the epicardial right ventricular action potential.
The R1192Q mutation had no effects on steady state activation
(not shown) but significantly accelerated decay of the sodium
current over a wide range of activation potentials (Fig. 9A–C).
Steady state inactivation of A735V was unchanged compared
to WT, displaying a V1/2 of –68.5 ± 0.2 and –68.4 ± 0.1 mV,
respectively. The R1192Q mutation produced a small but
significant positive shift of steady state inactivation (P < 0.05)
yielding a V1/2 of –64.5 ± 0.2 mV. The protocol consisted of
500 ms conditioning pulses ranging from –140 to –60 mV in
5 mV increments followed by a test pulse to 10 mV. The
holding potential was –90 mV.
Recovery from inactivation was significantly slower for
A735V channels than for WT (Fig. 10), an effect similar to that
Figure 3. Illustration of the SUNDS mutations within the cardiac sodium channel SCN5A, as well as the locations of mutations previously identified in Brugada
syndrome and LQTS patients.
340 Human Molecular Genetics, 2002, Vol. 11, No. 3
described previously for other Brugada syndrome-related
mutations (9,10) but was not changed by the R1192Q mutation
(data not shown).
Sudden cardiac death commonly results from ventricular
arrhythmias, but the underlying mechanisms responsible for
these tragic events are only now being unravelled. Over the
past decade, two disorders in which ventricular arrhythmias
play a central role in patient outcome, the long-QT (LQTS) and
Brugada syndromes, have been characterized at the genetic
level (16). In both instances, surface ECG abnormalities which
could be explained by the genetic defects and resultant physio-
logic derangements, are notable. In LQTS, seven genetic loci
have been identified (11–13,17) and the genes for six of these,
all ion-channel-encoding genes, have been described (14). Five
of these genes (LQT1, LQT2, LQT5, LQT6 and LQT7) encode
potassium channel proteins (KVLQT1, minK, HERG, MiRP1
and Kir2.1, respectively) whereas one, LQT3, encodes the
cardiac sodium channel gene SCN5A (16). Similarly, Brugada
syndrome has been shown to result from mutations in SCN5A,
but the biophysical properties of mutant sodium channels
differ in patients with LQTS (LQT3) (16,18,19) versus those
with Brugada syndrome (9,10). Because SUNDS is characterized
by ventricular tachyarrhythmias (VT/VF), has surface ECG
abnormalities similar to those seen in Brugada syndrome, and
is associated with nocturnal sudden death (1–4) like that
described in some LQT3 patients (20), we screened patients
and their families for SCN5A abnormalities. In two families
with autosomal dominant inheritance, and one sporadic case,
mutations were identified, all in the N-terminal portion of the
SCN5A protein. Although we screened all families for
mutations in the other ion channel candidate genes described
above, no mutations other than in SCN5A were identified. In
addition to LQTS, Brugada syndrome, and now SUNDS,
mutations in SCN5A have also been found to cause progressive
conduction system disease (Lev-Lenegre syndrome) (21),
isolated conduction system disease (22), and sudden infant
death syndrome (SIDS) (9,21,23,24). Interestingly, death most
commonly occurs during sleep in all of these disorders,
suggesting a common mechanism. Furthermore, mutations in
SCN5A have been observed in patients with features of both
LQTS and Brugada syndrome suggesting variable clinical
presentations with mutations in the same gene (7,25). In family
M033, the dizygotic twins presented during infancy, with one
child dying of apparent SIDS, while his brother survived due to
resuscitation of his ventricular tachyarrhythmias. Of note,
previously reported Brugada mutations have been shown to
cause the sodium channel to enter an intermediate inactivation
state from which it recovers more slowly (26). Whereas this
would lead to a reduction in INa at relatively rapid rates, it is not
clear how this mechanism could contribute to the Brugada
phenotype and arrhythmogenic substrate, both of which are
Biophysical analysis of the R367H mutation demonstrated
non-functional channels with no expression of inward currents.
To confirm that this was not due to a secondary mutation, the
entire cDNA cassette was sequenced (data not shown) and the
integrity of the RNA injected was assessed on a denaturing gel
(data not shown). The A735V mutation resulted in the expression
of robust INa, with steady state activation shifted towards more
positive potentials, similar to that described previously for the
Brugada syndrome at physiologic temperature (10). These
channels displayed a slower recovery from inactivation than
WT, again similar to that described previously for T1620M
(9,10,27,28). The shift in A735V steady state activation leads
to a shorter time to peak current as well as to reduced INa during
a time and voltage corresponding to phase 1 of the right
ventricular action potential. These two effects would be
expected to reduce the contribution of INa during phase 1,
leaving the transient outward current (Ito) unopposed.
The R1192Q mutation similarly shifts the balance of current
towards Ito by accelerating the decay of INa. This effect is
similar to that observed for two other Brugada mutations:
T1620M (10) and L567Q (29,30).
Although different mechanisms are involved in A735V and
R1192Q, the reduced density of INa present during activation of
Ito (phases 0 and 1 of the action potential) can lead to an
accentuation of the action potential notch and loss of the action
Figure 4. Mutation detection in Family M032. (A) SSCP analysis demonstrates
an abnormal conformer (arrow) in affected individuals of this family in exon 14
of SCN5A. Note the pedigree which identifies three affected males. (B) DNA
sequence analysis of the reverse primer product identifies a C→T substitution
in exon 14 of SCN5A which results in substitution of the WT alanine by valine
at codon 735 (A735V). (C) DHPLC confirms an abnormal band migration in
Human Molecular Genetics, 2002, Vol. 11, No. 3
potential dome. This rebalancing of net current present during
phase 1 of the action potential can lead to loss of the action
potential dome in cells possessing a prominent Ito (e.g. right
ventricular epicardium) but not those largely devoid of an Ito
(e.g. endocardium or left ventricular epicardium). Transmural
or transepicardial dispersion is likely to develop as a
consequence, leading to the ST-segment elevation in the right
precordial leads (V1–V3) on surface ECG recordings and VT/VF,
the hallmarks of both Brugada syndrome and SUNDS (31).
We conclude that Brugada syndrome and SUNDS represent
the same autosomal dominant familial disorder, suggesting this
disorder occurs worldwide. Both SUNDS and Brugada syndrome
can result from mutations in the cardiac sodium channel
SCN5A that cause loss of channel function but, like Brugada
syndrome, mutations have only been identified in a proportion
of the probands studied. Furthermore, this work suggests that
SUNDS, Brugada syndrome, SIDS, LQTS and conduction
system disease are allelic disorders, if not the same disease
with variable penetrance and variable modifiers. The methods
used to study these patients (PCR, SSCP and DHPLC), which
are sensitive for the detection of point mutations or small
deletions, could miss large gene deletions. Furthermore,
promoter mutations resulting in aberrant gene expression
cannot be discounted in patients testing negative for mutations.
However, it appears likely that there is genetic heterogeneity in
both of these disorders, with mutations in other ion channel
genes or proteins involved in phase 1 repolarization of the
ventricular action potential either directly or through modulation
of ion channel function. Incomplete penetrance of these mutations
could result in asymptomatic carriers. It is likely that provocation
studies using ajmaline, flecainide or procainamide could be
Figure 5. Mutation detection in family M033. (A) DHPLC identifies an abnormal band migration in exon 20 of SCN5A. (B) Sequence analysis demonstrates a
G→A substitution nucleotide 3727, which results in substitution of arginine by glutamine at codon 1192 (R1192Q).
Figure 6. Heterologous expression of the A735V and R367H channels in
Xenopus laevis oocytes. (A) WT SCN5A. (B) Mutant A735V. (C) Mutant
R376H. (D) Endogenous currents. All currents were recorded using a 20 ms test
pulse preceded by a 200 ms conditioning pulse to –120 mV from a holding
potential of –90 mV. Current traces shown in (A–D) were elicited by potential
steps from –40 to +10 mV in 5 mV increments.
342 Human Molecular Genetics, 2002, Vol. 11, No. 3
useful in evaluation of asymptomatic SUNDS family
members, as has been shown for Brugada syndrome (32,33).
Finally, similar therapeutic options should be considered for
both disorders; currently, implantation of an automatic implantable
cardioverter defibrillator is an option to consider in these
MATERIALS AND METHODS
Kindreds and sporadic cases with clinical evidence of SUNDS
were enrolled from medical clinics in Japan and Thailand. The
phenotype of each family member was characterized by a
previous history of sudden death in probands or in a family
member, no demonstrable structural heart defects on
echocardiogram or prolonged QT interval on surface ECG, and
an ECG pattern of ST-segment elevation in leads V1–V3, with
or without RBBB. Detailed family history was obtained from
all probands and their families, along with the history of all
clinical events. All enrolled individuals underwent evaluation
by surface ECG and Holter monitor. When clinically indicated,
an electrophysiology study was performed. In all instances,
written informed consent was obtained.
DNA mutation analysis
Genomic DNA was extracted directly from blood or from
lymphoblastoid cell lines established from blood, using
standard protocols (34). Genomic DNA samples were
amplified by PCR using primers designed to amplify across the
entire sequence of all known LQTS and Brugada syndrome genes
(KvLQT1, HERG, SCN5A, minK, MiRP1) in an exon-by-exon
manner (11–13). PCR products were analyzed by SSCP analysis
(9,34) or by DHPLC using a WAVE DNA Fragment Analysis
System (Transgenomic, Omaha, NE) (35). Normal and aberrant
SSCP conformers were cut directly from dried gels and eluted in
distilled water (65°C for 30 min) and then re-amplified. For
samples giving abnormal DHPLC peaks, the genomic DNA
was re-amplified directly. PCR products were sequenced,
using Big Dye Terminator chemistry and an ABI-310 automated
sequencer (PE Biosystems, Foster City, CA), according to the
manufacturer’s instructions. 100 control patients of Asian
descent were used to exclude the likelihood of the
abnormalities being benign polymorphisms.
Mutant SCN5A channel cDNAs were prepared by site-directed
mutagenesis of the plasmid pcDNA–SCN5A which contains
SCN5A cDNA cloned into pcDNA3.1+ (Invitrogen, Carlsbad,
CA). To create the R367H mutant, the ‘Megaprimer’ method of
mutagenesis was used. In brief, a 100 bp fragment containing the
mutated sequence was amplified from pcDNA–SCN5A using the
primers SCN5A–AgeI (AGGGCTACCGGTGCCTAAAGGCAG)
and SCN5A–R367H (CGTCATCAGGTGGAACACTG). After
gel purification this was used as a ‘megaprimer’ along with
SCN5A–NheI (CCGAGTCGTTCTTGCCAAAGAGCTG), to
amplify a product of 1587 bp from pcDNA–SCN5A. This
fragment was cloned back into pcDNA–SCN5A by substitution
of the AgeI–NheI fragment (nucleotides 1017–2536 of SCN5A
A similar approach was used for constructing the A735V
mutant. To create the ‘megaprimer’, pcDNA–SCN5A was
amplified using the primers SCN5A–A735V (CACTCTTCAT-
GGTGCTGGAG) and SCN5A–NheI. The 408 bp PCR
megaprimer was then used with SCN5A–AgeI to amplify the
1587 bp product, which was cloned in pcDNA–SCN5A as
above. Mutant clones were identified and characterized by
DNA sequencing with SCN5A–AgeI or SCN5A–NheI primers,
as described above.
Oocyte preparation, RNA injection and electrophysiology
Xenopus laevis were anesthetized and an incision was made in
the lower abdomen of the frogs to remove the ovarian lobes.
The oocytes were freed from the lobes and digested for 1–2 h
with 2 mg/ml collagenase (201 U/mg; Gibco BRL, Gaithersburg,
MD) to remove the follicular membrane. Healthy stage IV and
V oocytes were selected for RNA injection on the next day.
In vitro transcription of WT and mutant SCN5A cDNAs was
performed using an mMessage mMachine kit (Ambion,
Austin, TX). The RNA solution was diluted to the desired
concentration (250 or 500 ng/µl) in sterile 100 mM KCl and
Figure 7. Steady state activation of the WT and A735V mutant channels.
(A) Current–voltage relationship (I–V) of WT (squares, n = 6) and A735V
(circles, n = 8) obtained from current recordings as illustrated in Figure 5. The
peak current of A735V was significantly shifted from –10 to –5 mV when
compared to WT. (B) Steady state activation was obtained by normalizing the
chord conductance at each test potential of the I–V relationship to the maximal
(slope) conductance. Data ± SEM were fitted to a Boltzmann distribution
function (solid line) and yielded mid-activation potentials of –24.9 ± 0.4 mV
(n = 8) and –18.2 ± 0.2 mV (n = 11) for WT and A735V, respectively.
Human Molecular Genetics, 2002, Vol. 11, No. 3
Figure 8. Effects of the steady state activation shift on the current decay of A735V in a range of potentials corresponding to the end of phase 1 of the action
potential. (A) Current traces from WT and A735V during a test pulse to –30 mV were normalized to their respective maximal currents and superimposed. Although
the two current traces have the same time constants for decay kinetics, the contribution of A735V is significantly reduced over a time course of 4 ms when compared
to WT due to the 7 mV shift in steady state activation between the two channels (Fig. 6). (B) Current traces, as described in (A), obtained at –25 mV. (C) Maximal
peak currents of representative WT and A735V recordings are superimposed (WT, –15 mV; A735V, –5 mV). (D) Relative current values as described in (A–C) were
measured 3 ms after the start of the test pulse (dotted line) and plotted as a function of the test potential for WT (squares) and A735V (circles). ***P < 0.001, *P < 0.05 for
values at the same potential, ‡P < 0.05 between the marked values, each corresponding to the location of the maximal peak current as shown in (C).
Figure 9. R1192Q mutation accelerates fast inactivation of the sodium channel. (A) Representative currents recorded from WT (dotted line) and mutated channels
(R1192Q, solid line) following a pulse to –10 mV from a holding potential of –120 mV. Currents were normalized to their respective peak values (R1192Q, 4.7 µA;
WT, 5.6 µA). The dashed line 3 ms after the beginning of the pulse indicates the timing of peak transient outward current. 0 indicates the baseline level. (B and C) Current
decay was fitted to a sum of two exponentials and the fast and slow time constants of each parameter were plotted against the membrane potential. In (B), values
were statistically significant only in the range specified by the line labeled with an asterisk (P < 0.05). In (C), all values were statistically different (P < 0.01 except
at –10 mV). Number of cells tested: WT, 8; R1192Q, 12. (D) The R1192Q mutation significantly shifted the steady state mid-inactivation potential by +5 mV but
A735V had no effect on the availability of the channels. Data ± SEM were fitted to a standard Boltzmann distribution function (solid line). Number of cells tested:
WT, 11; A735V, 8; R1192Q, 18.
344 Human Molecular Genetics, 2002, Vol. 11, No. 3
46 nl of the cRNA solution was injected in each oocyte using a
Nanojet automatic oocyte injector (Drummond Instruments,
Broomall, PA). Once injected, the eggs were stored at 17°C in
SOS solution containing 100 mM NaCl, 2 mM KCl, 1.8 mM
CaCl2·2H2O, 1 mM MgCl2·6H2O, 5 mM HEPES, 2.5 mM
pyruvic acid, pH 7.6, supplemented with 100 µg gentamicin,
100 U/ml penicillin + 100 µg/ml streptomycin, under slow
Whole-cell currents were recorded from Xenopus oocytes
using the conventional two-microelectrode voltage-clamp
technique (10). Briefly, bevelled microelectrodes were
plugged with a 1.5% agarose solution containing 3 M KCl,
10 mM HEPES and 10 mM EGTA, pH 7.4 and back-filled
with 3 M KCl to give a resistance of 0.2–0.5 MΩ. Oocytes
were placed in a chamber and perfused with an external
solution containing 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
140 mM NaOH, 10 mM HEPES, 10 mM glucose, pH 7.4.
Currents were amplified by a Warner oocytes clamp (OC-725A),
low-pass filtered at 3 kHz (–3 dB, 4 pole Bessel filter,
Wavetech, Model 432). Data acquisition and analysis was
performed with pCLAMP 6 (Axon Instruments, Foster City,
CA). Currents were recorded at room temperature, and
experiments in which the holding current was >200 nA at a
holding potential of –90 mV were excluded from analysis.
The authors are grateful to Melba Koegele, Yue-Sheng Wu,
Stacy Scicchitano and Elena Burashnikov for expert technical
assistance. This work was funded by NIH grants RO1
HL62570 (J.A.T.), HL 47678 (C.A.) and HL 59449 (R.D.) and
by the Masons of the states of New York and Florida.
Jeffrey A.Towbin, MD is supported by the Texas Children’s
Hospital Foundation Chair in Pediatric Cardiovascular
Research. Neil E.Bowles, PhD is supported by grants from the
American Heart Association (Texas Affiliate and National).
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Figure 10. Recovery from inactivation of WT and A735V mutant channels. (A) Recovery from inactivation was measured using a standard double pulse protocol
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Human Molecular Genetics, 2002, Vol. 11, No. 3
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