Neuron, Vol. 34, 877–884, June 13, 2002, Copyright 2002 by Cell Press
Molecular Basis of an Inherited Epilepsy
2001; Sugawara et al., 2001b; Wallace et al., 2001b).
A closely related inherited epilepsy, severe myoclonic
epilepsy of infancy, has also been associated with
SCN1A mutations (Claes et al., 2001).
Throughout the central nervous system, voltage-
gated sodium channels are critical for the generation
tant pharmacological targets for anticonvulsant agents
(Catterall, 1999; Macdonald and Greenfield, 1997). The
pathological role of sodium channel mutations in epi-
lepsy is not well understood. Therefore, functional char-
nel alleles may shed new light on the molecular basis
of GEFS? specifically, advance our understanding of
ful for the design of new therapeutic interventions. Al-
though two groups have previously investigated the
functional consequences of GEFS? SCN1A mutations
2001; Spampanato et al., 2001), no studies have exam-
ined mutations in the authentic human SCN1A channel
in the presence of ?1 and ?2 subunits.
We report here the successful cloning and functional
characterization of human SCN1A along with electro-
physiological experiments demonstrating biophysical
disturbances conferred by three distinct GEFS? muta-
tions. Our findings indicate that GEFS? is caused by a
defect in sodium channel inactivation that leads to a
persistent inward current during sustained depolariza-
tions. This defect will likely cause prolonged neuronal
depolarization leading to increased firing frequency and
Christoph Lossin,1,2Dao W. Wang,1,4
Thomas H. Rhodes,1Carlos G. Vanoye,1,3
and Alfred L. George, Jr.1,2,3,4,5
1Division of Genetic Medicine
2Center for Molecular Neurosciences
3Department of Medicine
4Department of Pharmacology
Nashville, Tennessee 37232
Epilepsy is a common neurological condition that re-
flects neuronal hyperexcitability arising from largely
unknown cellular and molecular mechanisms. In gen-
eralized epilepsy with febrile seizures plus, an autoso-
mal dominant epilepsy syndrome, mutations in three
genes coding for voltage-gated sodium channel ? or
?1 subunits (SCN1A, SCN2A, SCN1B) and one GABA
receptor subunit gene (GABRG2) have been identified.
Here, we characterize the functional effects of three
mutations in the human neuronal sodium channel ?
subunit SCN1A by heterologous expression with its
known accessory subunits, ?1 and ?2, in cultured
mammalian cells. SCN1A mutations alter channel in-
activation, resulting in persistent inward sodium cur-
rent. This gain-of-function abnormality will likely en-
hance excitability of neuronal membranes by causing
prolonged membrane depolarization, a plausible un-
derlying biophysical mechanism responsible for this
inherited human epilepsy.
Idiopathic epilepsy is a common, paroxysmal, and clini-
cally heterogeneous neurological disorder with incom-
pletely understood cellular mechanisms (Browne and
Holmes, 2001; McCormick and Contreras, 2001; Shin
and McNamara, 1994). While many factors contribute
to the pathogenesis of epilepsy, a subset of familial
epilepsy syndromes are “channelopathies,” inherited
conditions caused by mutations in genes encoding vari-
ous voltage-gated and ligand-gated ion channels (Ber-
kovic,2001; Meisleretal., 2001).Among thesedisorders
are genetically distinct forms of generalized epilepsy
with febrile seizures plus (GEFS?), an autosomal domi-
nant syndrome characterized by febrile seizures and
various types of afebrile seizures (Scheffer and Ber-
kovic, 1997; Singh et al., 1999). GEFS? has previously
been linked to mutations in genes encoding voltage-
gated sodium channel ? (SCN1A, SCN2A) (Escayg et
et al., 1998) subunits and one GABA receptor subunit
(GABRG2) (Baulac et al., 2001; Wallace et al., 2001a).
Mutations in SCN1A represent the most frequent cause
of the disease (Abou-Khalil et al., 2001; Escayg et al.,
Cloning and Functional Expression
of Human SCN1A
The complete coding region (6030 bp) of human SCN1A
with 873 bp of the 3? untranslated region was deduced
as four overlapping cDNAs using reverse-transcriptase
polymerase chain reaction (RT-PCR) cloning as defined
in Experimental Procedures. The predicted 2009 amino
acid peptide exactly matches the sequence reported
by Escayg et al. (2000) (GenBank accession number
P35498). Two probable splice variants were also identi-
fied through sequencing of independently isolated
splice donor sequences contained within exon 11 and
produce in-frame deletions of 33 and 84 nucleotides
(encoding a portion of the cytoplasmic region between
ant form) in a mammalian expression plasmid was tran-
siently transfected into tsA201 cells along with plasmids
coupled to distinct reporter genes (CD8 antigen and
green fluorescent protein, respectively). Transfected
cells expressing both reporters and exhibiting voltage-
Figure 1. Functional Characterization of WT-
SCN1A in tsA201 Cells
(A) Typical current tracings from a tsA201 cell
transiently transfected with WT-SCN1A, h?1,
and h?2 recorded at various test potentials
between ?80 and ?50 mV (holding potential
was ?120 mV).
(B) Current-voltage relationship for WT-
SCN1A. Whole-cell currents were normalized
to cell capacitance (n ? 8).
(C) Voltage dependence of sodium channel
availability and activation. The voltage de-
pendence of sodium channel availability
(steady-state inactivation) was obtained us-
ing a standard double-pulse protocol indi-
cated as an inset. Half-maximal inactivation
was reached at ?67.5 ? 2.3 mV with a slope
factor of ?6.2 ? 0.3 (n ? 9). Half-maximal
activation occurred at ?26.4 ? 2.3 mV with
a slope factor of 7.1 ? 0.2 (n ? 8).
(D) Time course of recovery from inactivation
recovery from inactivation is shown as an in-
set. The time constants and fractional ampli-
tudes (given in parentheses) were as follows:
?f? 6.4 ? 1.3 ms (71% ? 3%), ?s? 263 ? 36
ms (29% ? 3%), n ? 9.
Figure 1 illustrates the biophysical behavior of recom-
binant human wild-type SCN1A (WT-SCN1A) coex-
ing and inactivating voltage-dependent inward currents
were observed in response to depolarizing test poten-
tials and were generally robust (typical peak current
amplitude range1–5 nA;Figure 1A).Endogenous inward
currents larger than 0.2 nA were never observed in non-
transfected cells. Peak activation of sodium current oc-
curred at ?10 mV (Figure 1B), and the expressed cur-
rents were highly tetrodotoxin (TTX) sensitive (data not
shown). Voltage-dependent channel availability and ac-
tivation were half-maximal at ?67.5 ? 2.3 and ?26.4 ?
tion following a 500 ms depolarization exhibited two
exponential components (Figure 1D), with a predomi-
nant fast (time constant, ?f? 6.4 ? 1.3 ms, 71% ? 3%)
and smaller slow component (?s? 263 ? 36 ms, 29% ?
3%). All of these properties are consistent with voltage-
gated sodiumchannels and closelyresemble previously
ties (Reckziegel et al., 1998; Sah, 1995).
application of 10 ?M TTX, thus excluding a leak current
(Figure 2D). The size of the noninactivating current var-
ied among the mutants, but was largest in R1648H-
0.2% ? 0.1%, n ? 4; R1648H, 4.2% ? 0.6%, n ? 5, p ?
0.0005 versus WT-SCN1A; T875M, 1.5% ? 0.2%, n ?
4, p ? 0.001 versus WT-SCN1A; W1204R, 0.9% ? 0.2%,
n ? 8, p ? 0.01 versus WT-SCN1A). A characteristic
displayed by noninactivating sodium channels is the
inappropriate activation of inward current during a slow
depolarization. This feature was demonstrated by com-
paring the responses of WT-SCN1A to R1648H using
the voltage-ramp protocol illustrated in Figure 3. Slow
membranedepolarization triggereda significantlylarger
inward current in cells expressing R1648H than in WT-
SCN1A-expressing cells (maximal ramp current divided
by the peak transient current ? 100 [mean ? SEM]: WT-
SCN1A, 0.3 ? 0.08%; n ? 6 versus R1648H, 1.9 ? 0.2%;
n ? 5, p ? 0.0001).
This inactivation disturbance was previously not ob-
served when similar GEFS? mutations were examined
in the human skeletal muscle sodium channel (Alekov
et al., 2000, 2001) or the rat SCN1A ortholog (Spampa-
nato et al., 2001), suggesting that species and isoform-
related variables may be important for revealing this
mutant sodium channel phenotype. Furthermore, the
absence of persistent sodium currents in the work re-
ported by Spampanato et al. (2001) may relate to abnor-
mal inactivation properties commonly observed for re-
combinant neuronal and muscle sodium channels
man et al., 1990; Zhou et al., 1991).
Mutant sodium channels expressed similar current
density as WT-SCN1A (Figure 4A) and exhibited no sig-
nificant differences in the voltage dependence of inacti-
vation time constants (Figure 4B). Other biophysical
properties of the mutant sodium channels grossly re-
sembled those of WT-SCN1A, with three exceptions. A
significant depolarizing shift in the voltage dependence
Epilepsy-Associated SCN1A Mutants
mutants using recombinant human SCN1A sodium
channels. Figures 2A–2C illustrate typical whole-cell re-
cordingsobtained fromcells expressingT875M, W1204R,
or R1648H. All three mutants exhibited robust, rapidly
activating and inactivating inward currents in response
ination of all recordings revealed the presence of nonin-
activating inward current in the mutants that was not
observed in WT-SCN1A. This noninactivating current
was evident during a longer test depolarization (100 ms)
and could be reversibly and completely blocked by the
Molecular Basis of GEFS?
Figure 2. Whole-Cell Recordings of Mutant
Typical current tracings from transiently
transfected tsA201 cells expressing SCN1A
mutants R1648H (A), T875M (B), and W1204R
between ?80 and ?50 mV stepped from a
holding potential of ?120 mV. In all experi-
ments, h?1 and h?2 were coexpressed. (D)
Representative WT-SCN1A, R1648H, T875M,
and W1204R TTX-sensitive sodium currents.
Sodium current was elicited by a 100 ms de-
polarization from ?120 to ?10 mV. TTX-sen-
sitive currents were obtained by digital sub-
traction of sodium currents recorded before
and after TTX addition. Peak sodium currents
were normalized. Zero-current level is indi-
cated by a dotted line. The inset shows an
expanded y axis scaled to emphasize the rel-
ative proportion of noninactivating current.
of channel availability was exhibited by T875M, and a
significant hyperpolarizing shift of channel activation
was observed for W1204R (Figure 4C). There was also
an enhancement of the slow component of recovery
from inactivation observed for T875M and R1648H (Fig-
ure 4D). The most consistent defect observed for all
three mutant sodium channels was the presence of a
noninactivating current component.
R1648H mutant to resolve single-channel behavior. Fig-
ures 5A and 5B illustrate representative patch clamp re-
nels exhibited predominantly early, short-lived (? 1 ms)
openings followed by infrequent late openings and rare
short bursts of reopenings. By contrast, mutant sodium
channels exhibited a much higher probability of late
openings occurring throughout 200 ms test depolariza-
tions. Multiple late openings were seen in all sweeps
recorded from mutant sodium channel patches. Esti-
potential was not different between alleles (WT-SCN1A,
16.6 ? 0.7 pS [n ? 7] versus R1648H, 17.3 ? 0.5 pS
[n ? 5]; test potential was 0 mV). Ensemble average
5C) closely resembled the whole-cell data presented
in Figures 1 and 2. These data indicate a significant
inactivation defect present in R1648H channels that ex-
plains the disturbance in sodium channel behavior ob-
served in whole-cell recordings (Figure 2D).
We recorded sodium currents in excised outside-out
patches from cells expressing either WT-SCN1A or the
We have extensively investigated the biophysical prop-
erties ofthree distinct GEFS? mutationsusing recombi-
nant human SCN1A coexpressed heterologously in cul-
tured mammalian cells with human accessory subunits
?1 and ?2. Because all essential molecular elements
necessary for assembly of human neuronal sodium
channels (Catterall, 1992) were provided, our experi-
ments have a high likelihood of revealing the true func-
tional defect responsible for this disease. Using this
approach, we also circumvented potential concerns re-
garding the molecular context of previous characteriza-
tions of GEFS? mutations in nonhuman or nonneuronal
sodium channels expressed in the absence of one or
both ? subunits (Alekov et al., 2000, 2001; Spampanato
et al., 2001). Such variability in experimental conditions
may explain critical differences between our observa-
tions and those of other investigators.
Our data demonstrate a clear defect in channel inacti-
vation exhibited by the GEFS? mutations T875M,
Figure 3. Responses of WT-SCN1A and R1648H to Ramp Depolar-
Cells were initially voltage clamped to a holding potential of ?120
mV to assure all sodium channels were available. The membrane
potential was slowly ramped from ?120 mV to ?40 mV over 8 s (20
mV/s). Traces represent TTX-sensitive currents obtained by digital
subtraction of sodium currents recorded before and after TTX (10
?M) addition. The dashed line indicates the zero current level. A
representative experiment is illustrated (the peak transient sodium
currents were ?3.6 nA for WT-SCN1A and ?3.2 nA for R1648H).
Similar experimental results were observed in four cells for WT-
SCN1A and five cells for R1648H.
Figure 4. Biophysical
Mutant SCN1A Channels
(A) Current-voltage relationships of WT-
SCN1A, R1648H, T875M,
Whole-cell currents were normalized to cell
capacitance and plotted against the test po-
tential (n ? 8–19).
(B) Voltage dependence of fast inactivation
time constants for wild-type and mutant
channels. Symbol shapes are defined in the
inset of (A). Data representing fast and slow
as open and closed symbols, respectively.
Symbols representing wild-type data appear
with the opposite shading pattern.
(C) Voltage dependence of sodium channel
availability and activation (symbol definitions
are similar to those in the inset of [A], and the
The pulse protocol shown as an inset refers
to channel availability only. The membrane
potentials for half-maximal inactivation and
slope factors were as follows (values for WT-
SCN1A are given in the Figure 1 legend):
R1648H, ?69.1 ? 2.1 mV and ?6.5 ? 0.4, n ?
10; T875M, ?60.7 ? 1.1 mV (p ? 0.01 versus
WT-SCN1A) and ?5.9 ? 0.6, n ? 13; W1204R, ?72.0 ? 2.0 mV and ?6.9 ? 0.4, n ? 7. The activation curve was constructed as described in
the legend of Figure 1. Half-maximal activation occurred at the following potentials: R1648H, ?25.9 ? 1 mV (n ? 15); T875M, ?26.1 ? 1.2
mV (n ? 19); W1204R, ?29.6 ? 1.2 mV (n ? 9, p ? 0.005 versus WT-SCN1A). Slope factors of the mutants were not significantly different
from that of WT-SCN1A (data not shown).
(D) Recovery from inactivation. Data were acquired according to the pulse protocol shown as an inset (symbol definitions given in the inset
of [A]). The time constants and fractional amplitudes (given in parentheses) were as follows (values for WT-SCN1A are given in Figure 1
legend): R1648H, ?f? 3.1 ? 0.2 ms (60% ? 4%), ?s? 257 ? 36 ms (40% ? 4%), n ? 9 (p ? 0.05 for ?sand fractional amplitudes); T875M,
?f? 4.6 ? 0.5 ms (55% ? 5%), ?s? 680 ? 151 ms (45% ? 5%), n ? 10 (p ? 0.05 for ?sand fractional amplitudes); W1204R, ?f? 5.3 ? 0.5
ms (68% ? 2%), ?s? 244 ? 31 ms (32% ? 2%), n ? 8.
W1204R, and R1648H. The observation of a significant
persistent noninactivating current component in all
three mutants studied suggests that gain-of-function
(i.e., increased sodium conductance) may be responsi-
ble for seizure susceptibility in this syndrome. This be-
havior is reminiscent of the channel dysfunction associ-
ated with two other human sodium channelopathies:
hyperkalemic periodic paralysis and congenital long QT
syndrome. Mutations in genes encoding the muscle so-
dium channel (SCN4A) (Cannon, 2000) or the cardiac
festing as a small but significant noninactivating late
sodium current. Interestingly, at the tissue level, this
type of channel dysfunction may result in increased ex-
citability, giving rise to myotonia in skeletal muscle and
ventricular arrhythmia in the heart. In muscle, however,
persistent depolarization of the sarcolemma can also
cause widespread inactivation of normal sodium chan-
nels, leading to excitation failure and paralysis (Cannon
et al., 1993).
Our data provide an important insight into the patho-
physiology of sodium channel dysfunction in epilepsy.
facilitates neuronal hyperexcitability because of a re-
duced threshold for action potential firing. The noninac-
tivating currents we observed in cells expressing
GEFS? mutants may correlate with characteristic in-
terictal activity, known as paroxysmal depolarization
shifts, observed in epileptic foci and in vitro models of
and Ajmone-Marsan, 1964; Segal, 1991, 1994). Altered
sodium channel function could also lead to a number
of other secondary cellular consequences, such as in-
also contribute to neuronal hyperexcitability (Dudek et
al., 1998; Jefferys, 1995). Determining the precise cellu-
lar consequences of this defect in the nervous system
requires further study.
we observed for the three distinct GEFS? mutations
may potentially correlate with the severity of the pheno-
type, although genotype-phenotype relationships are
difficult to construct reliably at this point from the small
number of reported families. Other biophysical distur-
bances were also observed in the SCN1A mutants, but
none were exhibited uniformly byall alleles. For mutants
T875M and W1204R, differences in the voltage depen-
dence of inactivation and activation, respectively, are
consistent with a gain-of-function. However, the en-
hancements in the slow component of recovery from
inactivation observedfor T875M and R1648Hmay result
in reduced sodium current density during long depolar-
izations or repetitive stimulation. These additional func-
ences between individuals carrying different mutations,
but more work is needed to make these associations.
Genetic modifiers and environmental factors are also
likely to impact substantially on disease expression. In-
dividuals with GEFS? appear to have an intrinsic sei-
Molecular Basis of GEFS?
Figure 5. Single-Channel Recordings of WT-
SCN1A and R1648H
Representative single-channel traces re-
corded inoutside-out membranepatches ex-
cised from tsA201 cells transiently trans-
fected withWT-SCN1A (A)or R1648H(B) plus
h?1 and h?2 subunits. The number of chan-
nels for WT-SCN1A and ?11 channels for
R1648H by dividing the largest current peak
measured during 100 sweeps by the unitary
conductance. Channel openings are shown
as downwarddeflections andsolid horizontal
lines indicate zero-current level. Channel ac-
tivity was measured for 200 ms at 0 mV from
a holding potential of ?100 mV. (C) Ensemble
average currents for WT-SCN1A and R1648H
generated from single-channel data. Four
independent experiments for each channel
ity (Po) obtained by dividing the ensemble av-
erage current by the unitary current (?1 pA)
and the number of channels per patch.
and Berkovic, 1997; Singh et al., 1999). Fever appears
to be an important nongenetic factor in the triggering
of seizures in GEFS?. At this time, we chose not to
ity between wild-type and mutant SCN1A alleles be-
cause of a variety of technical issues (increased thermal
noise, excessively rapid gating kinetics, channel run-
down) that make recordings of voltage-gated sodium
channels at physiologicaltemperatures unreliable. Also,
seizure susceptibility in GEFS? is not strictly linked to
fever, and most forms of febrile seizures occur in the
absence of known sodium channel defects.
As with many other epilepsy syndromes, GEFS? pa-
tients exhibit multiple typesof seizures, including partial
andgeneralized forms(Abou-Khaliletal., 2001;Scheffer
and Berkovic, 1997; Singh et al., 1999; Sugawara et al.,
2001b). Such pleomorphism is difficult to explain solely
on the basis of the sodium channel inactivation defect
observed in our studies. We speculate that the underly-
ing sodium channel dysfunction predisposes GEFS?
patients to seizures at an early age. Neuronal injury re-
sulting secondarily to convulsions may then evoke
pathological structural changes and chronically reduce
seizure threshold (McCormick and Contreras, 2001).
by early pharmacological intervention that specifically
targets the underlying sodium channel dysfunction. As
persistent neuronal sodium currents may be a common
2002), further studies of the pharmacology of mutant
SCN1A channels may provide an in vitro model system
specifically target this abnormal activity.
Molecular Cloning of Human SCN1A cDNA
The human SCN1A open reading frame (ORF) was predicted by
comparing the orthologous rat coding sequence (GenBank acces-
sion number NM_030875) to the human genomic sequence using
the program BLASTN (http://www.ncbi.nlm.nih.gov/blast). Twenty-
six exons were identified, ordered, and assembled into a 6030 bp
ORF and 873 bp 3? untranslated region (3?UTR). Based on this pre-
diction, four sets of PCR primers (AF: 5?-GTTTCTTGCGGCCGCATG
GAGCAAACAGTGCTTGTACCA-3?, AR: 5?-GTGT CTTTCCCTTCAAT
CTTTATGTCCAATCATACAGCAGA-3?, CR: 5?-GTGTCTTGGCTTAC
TGTTGAGAATGGG-3?, DF: 5?-GTTTCTTACGCCATTATTATTTTA
CCA-3?, DR: 5?-GTGTCTTGTCGACTCAAGGTCATCTCCCCTTTA-3?)
bral cortex cDNA (Clontech, Palo Alto, CA) was used as template
during hot-start PCR performed in 50 ?l reactions at 94?C for 5 min
followed by 35 cycles of 94?C for 1 min, 51?C–57?C for 1 min, and
72?C for 3–4 min, and a final cycle at 72?C for 5 min. Some reactions
required the addition of 10% (v/v) Q solution (Qiagen, Valencia, CA).
a combination of Taq and PwoI (20% v/v; Roche, Indianapolis, IN)
Reaction products were gel-extracted, then cloned into pCR2.1-
TOPO (Invitrogen, Carlsbad, CA), and selectively grown in TOP10,
INV?F? (Invitrogen), or STBL2 cells (Life Technologies, Grand Island,
by restriction fingerprinting, and sequenced using automated fluo-
rescent dye terminator chemistry. Polymerase errors were repaired
bysite-directedmutagenesis orbysubcloningfragments fromother
completely. A short poly-T region exhibiting a high spontaneous
mutation rate in the full-length construct was interrupted by the
introduction of two silent T to C mutations at ORF positions 1206
and 1209. All full-length constructs were propagated in STBL2 cells
grown at 30?C for ?48 hr.
CsF, 20 mM CsCl, 2 mM EGTA, 10 mM HEPES [pH 7.35, 310 mOsm/
kg]) was matched in pH and osmolality to the bath solution. Patch
pipettes were pulled from borosilicate glass (Warner Instrument,
Hamden, CT) with a multistage P-97 Flaming-Brown micropipette
puller (Sutter Instruments, San Rafael, CA) and fire-polished with a
Micro Forge MF 830 (Narashige, Japan). Patch pipettes for single-
channel studies were coated with Sylgard 184 (Dow Corning, Mid-
land, MI). Pipette resistance was 0.8–1.5 M? for whole-cell and ?4
M? for single-channel experiments. Cells were allowed to stabilize
for 10 min after establishment of the whole-cell configuration before
currents were measured. Recordings from cells exhibiting peak cur-
rent amplitudes less than 0.8 nA were excluded from analysis to
avoid potential endogenouschannel contamination. Cells exhibiting
very large whole-cell currents were also excluded if voltage control
was compromised. Whole-cell capacitance was assessed by inte-
grating the capacitive transient elicited by a 10 mV voltage step
from ?120 mV to ?110 mV with 10 kHz filtering. As a reference
electrode, a 2% agar bridge with composition similar to the bath
solution was utilized. Whole-cell currents were acquired at 20–50
kHz and filtered at 5 kHz. Single-channel current traces were ac-
quired at 10 kHz and filtered at 1 kHz.
Channel behavior was examined over a range of test potentials
(see figure insets for pulse protocols). Each voltage step was fol-
lowed by a 5 s recovery period at ?120 mV. Pulse generation, data
collection, and analyses were done with pCLAMP 7.0 or 8.1 (Axon
Instruments), Excel 97 (Microsoft, Seattle, WA), Origin 6.0 (Microcal,
Northampton, MA), and Sigma Plot 2000 (SPSS Science, Chicago,
ting the peak current against the test potential. Steady-state inacti-
current and plotted as open probability versus prepulse potential.
Data were fitted to a two-state Boltzmann equation:
SCN1A mutants T875M, W1204R, and R1648H were named ac-
cording tothe single-lettercode indicatingthe aminoacid exchange
and its position with respect to the starting methionine. Mutants
ful introduction of the mutations was monitored by digestion at
engineered silent restriction sites, and all mutant cDNAs (complete
coding regions) were resequenced fully before use in experiments.
1 ? C
e(x ? V1/2)/k? C,
Cell Culture and Transfection
Human tsA201 cells, a HEK-293 derivative stably transfected with
medium (DMEM) supplemented with 10% (v/v) fetal bovine serum
(Atlanta Biologicals, Norcross, GA), 2 mM L-glutamine, and penicil-
fied 5% CO2atmosphere at 37?C. Only cells from passages ? 13
Expression of SCN1A, ?1, and ?2 was achieved by transient plas-
mid transfection using Qiagen Superfect reagent. Approximately 6
?g of total DNA was transfected (plasmid mass ratio was ?:?1:?2 ?
h?1and h?2werecloned intoplasmidscontainingthe markergenes
CD8 (pCD8-IRES-h?1) or GFP (pGFP-IRES-h?2) along with an inter-
nal ribosome entry site. Cells were passaged 24 hr after transfection
and incubated 24 hr before their use in electrophysiology experi-
ments. Transfected cells were dissociatedby brief exposure to tryp-
sin/EDTA, resuspended in supplemented DMEM medium, and al-
lowed to recover for ?30 min at 37?C in 5% CO2. CD8 antibody-
covered microbeads (Dynabeads M-450 CD8, Dynal, Norway) sus-
pended in 200 ?l DMEM were added to the cell suspension and
gently shaken. In order to allow for patch excision in single-channel
studies, tsA201 cells were plated on glass coverslips pretreated
with CELL-TAK cell and tissue adhesive (Collaborative Biomedical
Products, Bedford, MA). Only cells positive for CD8 antigen and
GFP fluorescence were used for electrophysiological studies. Non-
transfected cells were used as negative controls.
where V1/2is the voltage at which 50% of the channels are inacti-
vated, k is the slope factor, and C is the steady-state asymptote.
Time constants (?) of inactivation were derived from the current
decay fitted to a single or a double exponential function:
f (t) ??
i ? 1
Ai· e?(t ? K)/?i? C,
where t is the time, A is the fraction of channels inactivating with
time constant ?i(?fand ?srepresent fast and slow time constants,
respectively), and K is the manually selected point of onset of expo-
nential macroscopiccurrent decay.Data fordetermining thevoltage
dependence of activation was derived from calculating conduc-
tances using the formula:
V ? Erev
where I(V) is the peak raw current at the clamping potential V, and
Erevis the estimated reversal potential. Conductances were normal-
ized to the maximal conductance between ?80 and ?20 mV and
fitted to the two-state Boltzmann equation:
f (x) ?
e(x ? V1/2)/k? 1,
where V1/2is the voltage at which half-maximal activation occurs,
and k describes the slope of the fit. Recovery from inactivation was
also examined by a two-pulse protocol. The peak current amplitude
during the test pulse was plotted as fractional recovery against the
recovery period by normalizing to the maximum current during the
conditioning, followed by fitting to a single or a double exponential
Electrophysiology and Data Analysis
of an inverted microscope with epifluorescence capability. After
in the whole-cell or excised, outside-out patch configuration of the
patch-clamp technique (Hamill et al., 1981) using Axopatch 200
series amplifiers (Axon Instruments, Union City, CA). Bath solution
(145 mMNaCl, 4mM KCl,1.8 mMCaCl2, 1mM MgCl2,10 mMHEPES
[pH 7.35, 310 mOsm/kg]) was continuously exchanged by a gravity-
driven perfusion system. The pipette solution (10 mM NaF, 110 mM
f (t) ??
i ? 1
Ai(1 ? e?t/?i),
where t is time, and Aidescribes the fraction of channels recovering
All experiments were performed at room temperature. Data are
Molecular Basis of GEFS?
shown as means ? SEM with the number of experiments provided
as n in the figure legends. Statistical comparisons were done with
the Student’s t test and differences were considered significant at
the p ? 0.05 level.
D., et al. (2000). Mutations of SCN1A, encoding a neuronal sodium
channel, in two families with GEFS?2. Nat. Genet. 24, 343–345.
Escayg, A., Heils, A., MacDonald, B.T., Haug, K., Sander, T., and
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Received: January 14, 2002
Revised: April 12, 2002
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