Divergent sodium channel defects in familial
Kristopher M. Kahlig*, Thomas H. Rhodes*, Michael Pusch†, Tobias Freilinger‡, Jose ´ M. Pereira-Monteiro§¶,
Michel D. Ferrari?, Arn M. J. M. van den Maagdenberg?**, Martin Dichgans‡, and Alfred L. George, Jr.*††‡‡
Departments of *Medicine and††Pharmacology, Vanderbilt University, Nashville, TN 37240;†Istituto di Biofisica, Consiglio Nazionale delle Ricerche,
16149 Genoa, Italy;‡Department of Neurology, Ludwig-Maximilians-University, 80539 Munich, Germany;§Instituto de Cie ˆncias Biome ´dicas
Abel Salazar, Universidade do Porto, 4099-002 Porto, Portugal;¶Servic ¸o de Neurologia, Hospital Geral de Santo Anto ´nio, 4099-001 Porto,
Portugal; and Departments of?Neurology and **Human Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
Edited by David E. Clapham, Harvard Medical School, Boston, MA, and approved May 8, 2008 (received for review December 13, 2007)
Familial hemiplegic migraine type 3 (FHM3) is a severe autosomal
dominant migraine disorder caused by mutations in the voltage-
gated sodium channel NaV1.1 encoded by SCN1A. We determined
the functional consequences of three mutations linked to FHM3
(L263V, Q1489K, and L1649Q) in an effort to identify molecular
defects that underlie this inherited migraine disorder. Only L263V
and Q1489K generated quantifiable sodium currents when coex-
pressed in tsA201 cells with the human ?1 and ?2 accessory
subunits. The third mutant, L1649Q, failed to generate measurable
whole-cell current because of markedly reduced cell surface ex-
pression. Compared to WT-NaV1.1, Q1489K exhibited increased
well as delayed recovery from fast and slow inactivation, thus
resulting in a predominantly loss-of-function phenotype further
demonstrated by a greater loss of channel availability during
repetitive stimulation. In contrast, L263V exhibited gain-of-
function features, including delayed entry into, as well as acceler-
ated recovery from, fast inactivation; depolarizing shifts in the
steady-state voltage dependence of fast and slow inactivation;
increased persistent current; and delayed entry into slow inacti-
vation. Notably, the two mutations (Q1489K and L1649Q) that
exhibited partial or complete loss of function are linked to typical
FHM, whereas the gain-of-function mutation L263V occurred in a
family having both FHM and a high incidence of generalized
epilepsy. We infer from these data that a complex spectrum of
NaV1.1 defects can cause FHM3. Our results also emphasize the
complex relationship between migraine and epilepsy and provide
further evidence that both disorders may share common molecular
epilepsy ? SCN1A ? NaV1.1 ? FHM3
estimated 30 million migraine sufferers in the United States
alone represent a significant public health burden. Genetic
factors are important determinants of migraine susceptibility,
and understanding the molecular basis of rare monogenetic
migraine syndromes may help unravel the pathophysiology of
more common forms of migraine (2).
Familial hemiplegic migraine (FHM) is a rare autosomal dom-
inant form of migraine with aura (MA) characterized by the
presence of transient motor weakness and other aura symptoms,
such as visual and speech disturbances (3). FHM probands also
have an increased incidence of typical MA attacks, suggesting that
FHM and MA may exhibit shared pathophysiology (4). Mutations
linked to FHM. FHM1 is caused by mutations in CACNA1A
encoding the pore-forming ?1-subunit of the neuronal calcium
channel, CaV2.1 (5), whereas mutations in ATP1A2 encoding the
catalytic ?2-subunit of the glial Na?/K?-ATPase in adult brain
cause FHM2 (6). Two missense mutations (Q1489K and L1649Q)
in SCN1A encoding the pore-forming ?-subunit of the voltage-
igraine is a common paroxysmal neurovascular disorder
with a lifetime prevalence of 14–18% worldwide (1). An
gated sodium channel NaV1.1 have been linked to FHM3 (7, 8).
Corresponding mutations engineered into the paralogous cardiac
NaV1.5 were observed to cause significant functional disturbances,
further supporting the pathogenicity of these alleles. At the time,
channel were not successful.
SCN1A mutations have been previously associated with various
(generalized epilepsy with febrile seizures plus) to debilitating
(severe myoclonic epilepsy of infancy). Functional studies of epi-
lepsy-associated NaV1.1 mutants have demonstrated either func-
channels (9–15). Among the subset of missense mutations charac-
terized, there are no general correlations between mutation loca-
tion in the sodium channel protein and the functional conse-
quences. Therefore, prediction of the functional effects of FHM3
mutations is not feasible without direct experimental evidence.
Here we determined the molecular basis of FHM3 by examining
the biophysical or biochemical properties of three FHM3 mutants
engineered in a recombinant human NaV1.1 channel. We studied
two previously published mutations (Q1489K and L1649Q) as well
as a third mutation (L263V) recently identified in an FHM family
in which the majority of mutation carriers also have epilepsy (42).
Our data revealed that FHM3 is associated with a wide range of
NaV1.1 defects, suggesting that multiple molecular mechanisms
may underlie FHM3. Furthermore, specific biophysical features
observed for L263V may also explain the higher prevalence of
epilepsy associated with this mutation.
In this study, we determined the functional consequences of three
FHM3 mutations engineered in human NaV1.1 to ascertain the
molecular defects underlying this disorder. Two of the FHM3
mutations we studied were reported previously (Q1489K and
L1649Q) (7, 8). Q1489K introduces a positive charge 11 residues
N-terminal to the Ile–Phe–Met motif within the cytoplasmic linker
between domain III (DIII) and DIV, a structure essential for fast
a conserved arginine (Arg-1648) in the S4 voltage sensor segment
Author contributions: K.M.K., T.H.R., M.P., T.F., J.M.P.-M., M.D.F., A.M.J.M.v.d.M., M.D.,
and A.L.G. designed research; K.M.K., T.H.R., and A.L.G. performed research; M.P., T.F.,
J.M.P-M., M.D.F., A.M.J.M.v.d.M., and M.D. contributed critical information enabling the
study; K.M.K., T.H.R., and A.L.G. analyzed data; and K.M.K., M.D.F., A.M.J.M.v.d.M., M.D.,
and A.L.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Hall, Vanderbilt University, 2215 Garland Avenue, Nashville, TN 37232-0275. E-mail:
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
July 15, 2008 ?
vol. 105 ?
no. 28 ?
glutamatergic synapse. Therefore, we anticipate the pathophysiol-
ogy of mutant NaV1.1 in FHM3 to reflect a more complex
mechanism. Genetically engineered animal models expressing mu-
tant NaV1.1 channels in specific neuronal subpopulations will be
invaluable to elucidate the role of dysfunctional NaV1.1 in FHM3.
Materials and Methods
Mutagenesis and Expression of Human NaV1.1 cDNA. Mutagenesis of recombi-
the complete ORF of each construct to exclude polymerase-induced errors and
channels was performed in human tsA201 cells (HEK293 cells stably expressing
the simian virus 40 large T antigen). Cells were grown in Dulbecco modified
penicillin/50 ?g/ml streptomycin in a humidified, 5% CO2atmosphere at 37°C.
?g of DNA was transfected at a plasmid mass ratio of 7:1:1 for ?1/?1/?2). The
DsRed (DsRed-IRES2-h?1) or EGFP (EGFP-IRES2-h?2) along with an internal ribo-
some entry site. Unless otherwise noted, all reagents were purchased from
Electrophysiology. Whole-cell voltage-clamp recordings were used to measure
the biophysical properties of WT and mutant NaV1.1 channels, as previously
(21–22°C), 24–48 h after transfection. Additional experimental details are pro-
vided in SI Materials and Methods.
Data analysis was performed by using Clampfit 9.2 (Axon Instruments), Excel
one-way ANOVA followed by a Tukey post hoc test in reference to WT-NaV1.1.
Cell Surface Biotinylation. Cellsurfacebiotinylationwasperformedasdescribed
previously with minor modifications for NaV1.1 detection (38–41). WT-NaV1.1
and L1649Q were epitope-tagged with a triple FLAG tag (3XFLAG) on the C
terminus to increase detection sensitivity. The presence of the 3XFLAG on WT-
not shown). Membrane impermeant sulfo-NHS-biotin reagent (Pierce Biotech-
against the FLAG epitope anti-FLAG M2 (mouse) at 1:15,000 (Sigma–Aldrich).
Immunoreactive bands were visualized by using horseradish peroxidase-
nology) directed against the primary antibody, ECL Plus (GE Healthcare Bio-
Sciences), and Hypersensitive ECL film detection. Densitometry of protein bands
was used to quantitate protein levels (ImageJ software, National Institutes of
Health). Normalization to the total was used to control for protein loading.
Additional experimental details are provided in the SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Melissa Daniels and Maria Jose ´ Melo e Castro
(Porto, Portugal) for valuable technical assistance. This work was supported by
National Institutes of Health Grant R37-NS032387 (to A.L.G.), an Epilepsy Foun-
dation postdoctoral fellowship (to K.M.K.), and National Institutes of Health
Postdoctoral Fellowship F32-NS055591 (to K.M.K.).
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www.pnas.org?cgi?doi?10.1073?pnas.0711717105Kahlig et al.