Supporting Information

Supporting Information 10.1073/pnas.0908359107 SI Methods reverse PCR primer using the BigDye Terminator Cycle Se- Subjects. This study includes a three-generation dominant quencing Kit v3.1 (Applied Biosystems), purified with Sephadex migraine family referred to Vall d’Hebron University Hospital, G-50 (Amersham Biosciences) using MultiScreen Plates (Milli- Barcelona. Seven affected and three healthy individuals were pore), and run in an ABI PRISM 3700 DNA analyzer (Applied directly interviewed and examined by one of the authors (A.M.), Biosystems). Sequence chromatograms were inspected manually following the guidelines of the International Classification of and analyzed with the SeqMan v3.6 software (DNASTAR, Inc). Headache Disorders of the International Headache Society (1). CACNA1A gene mutation (c.1360G > A; A454T) was con- For the genetic studies, the recruited control individuals and firmed by sequencing reverse DNA strand and HphI restriction their first-degree relatives lacked any history of recurrent or analysis of exon 11 PCR product. Restriction analysis was also disabling headache. used to check for the presence of the mutation in all of the members of the pedigree and in a group of 150 unrelated Linkage Analysis. Genomic DNA was isolated from consenting healthy Spanish individuals. The mutation identified was named subjects, 10 family members, and 150 healthy unrelated individuals using the QIAamp DNA Blood Maxi Kit (QIAGEN) according to Human Genome Variation Society guidelines (approved by Universitat de Barcelona’s Ethical Committee (http://www.hgvs.org/) using RefSeq entry NM_001127221 as a following the Helsinki Declaration). Eight microsatellite markers cDNA reference sequence, with nucleotide 237A (ATG) cor- (synthesized using sequence data from the GDB (http://www. responding to +1, and NP_001120693 as the protein reference gdb.org/) and CEPH (http://www.cephb.fr/) databases) flanking sequence. Both sequences correspond to the CACNA1A tran- the CACNA1A (D19S586, D19S1165, D19S714), ATP1A2 script variant 3. (D1S3466, D1S1595, D1S1677) and SLC1A3 (D5S426, D5S418) genes, and three SNPs (rs8191987, rs994399, rs1542484) located Voltage Protocols and Fittings. Recordings were obtained with a D- fi fi within the SCN1A gene were genotyped in all of the available 6100 Darmstadt ampli er (List Medical) ltered at 1 kHz [only family members to test potential linkage of migraine to these for short (ms) pulses] and corrected for leak and capacitive loci. The genetic distances (in cM) between the markers are currents using the leak subtraction procedure (P/8 for short pulses according to the Marshfield map (available at http://www. and P/4 for long pulses). Currents were acquired at 33 kHz (for marshfieldclinic.org/research/pages/index.aspx). The haplotypes short pulses) or 5 kHz (for long pulses). Series-resistance com- were constructed manually by assuming the minimum number of pensation ≥80% was employed. The pClamp8 software (Axon recombinations (Fig. S1). Instruments) was used for pulse generation, data acquisition, and PCR reactions were performed under the following conditions: subsequent analysis. 50 ng genomic DNA, 0.2 mM dNTPs, 2.5 pmol each primer, 2 mM Peak inward Ca2+ currents were measured from cells clamped MgCl2, 0.5 U AmpliTaq Gold DNA polymerase (Applied Bio- at –80 mV and pulsed for 20 ms from –60 mV to +70 mV in 5-mV × fi μ systems), and 1 PCR Gold buffer in a nal volume of 25 L. steps. The following modified Boltzmann equation (Eq. 1) was fi Ampli cation conditions were 10 min at 94 °C, 35 cycles with 1 fitted to normalized current voltage (I–V) to obtain the voltage fi min at 94 °C, 1 min at 56 °C, 1 min at 72 °C, and a nal extension dependence of activation step of 10 min at 72 °C. For the microsatellite markers, the PCR products were separated on a 6% acrylamide/bisacrylamide 19:1 I ¼ GmaxðV − VrevÞ=ð1 þ expð − ðV − V1=2;actÞ=kactÞÞ; [1] gel and visualized by silver staining (2). For the SNPs, we performed direct sequencing of the PCR products, following the where I is thepeak current, Gmax is themaximum conductance ofthe procedure indicated below. cell, V is the membrane potential, Vrev is the extrapolated reversal potential of ICa, V1/2, act is the voltage for half-maximal current Mutation Analysis. SCN1A and SLC1A3 were not considered activation, and kact is the slope factor of the Boltzmann term. disease-causing genes in this pedigree because several hemi- Time constant for activation (τact), deactivation (τdeact), and plegic patients did not share the same haplotype. Analysis of inactivation (τinact) were obtained from single exponential fits of markers flanking the ATP1A2 gene indicated that all of the af- ICa activation phase, tail currents obtained with 20-ms prepulse fected individuals shared at least part of the genomic segment to +20 mV (to maximally open the voltage-gated P/Q channels) including the gene. The analysis of markers around the CAC- and followed by test pulses between –80 mV and +10 mV (in 5 mV NA1A gene showed that individual I.2 transmitted the same steps) for 30 ms, and the inactivation phase of I during a 3-s pulse combination of alleles to probands II.3, II.5, II.6, III.1, and III.2 Ca from a holding potential of –80 mV to a test potential of +20 mV, (all displaying FHM or MA with tongue/facial paresthesia), and respectively. a different combination of alleles was passed to proband II.1 Time course of I recovery from inactivation (τ ) was who, like her mother, presents MA without sensorimotor Ca recinact tested, applying a second pulse of 50 ms to +20 mV at increasing symptoms. Accordingly, the 47 exons of the CACNA1A gene and ATP1A2 time intervals (1-106 s) after the inactivating prepulse. Normal- the 23 exons of the gene and their corresponding exon/ fi intron junctions, including splice sites and branch points, were ized peak ICa at different times was tted to a single exponential. PCR amplified (following a standard procedure similar to that Voltage dependence of steady-state inactivation was estimated describe previously) and sequenced from individual II.5 (hemi- by measuring peak ICa currents at +20 mV following 30-s steps to – plegic migraine) and a healthy individual in 42 and 16 in- various holding potentials (from 100 to +10 mV;). During the – dependent PCR products, respectively. The coding region of the time interval between test pulses (20 ms), cells were held at 80 CACNA1A gene was sequenced also in individual II.1 (migraine mV. ICa was normalized to maximal ICa and half-maximal voltage with visual aura). The PCR products were column purified using (V1/2, inact), and slope factor for steady-state inactivation (kinact) the GFX PCR DNA and Gel Band Purification Kit (Amersham was obtained by fitting the data to the following Boltzmann Pharmacia Biotech), sequenced from either the forward or the equation (Eq. 2): www.pnas.org/cgi/content/short/0908359107 1of7 ÈÀ = = − = : [2] Total Protein Extraction. Forty-eight hours after transfection, cells I Imax ¼ 1 1 þ exp½ðV V1=2; inactÞ kinactg were washed twice with PBS solution, and whole-cell extracts were obtained by resuspension with cell scraper in BNP40 lysis buffer [50 Alternatively, voltage dependence of steady-state inactivation was mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 1 mM evaluated from a 20-ms test pulse to +20 mV following a 30-s μ – – DTT, 5 mM EDTA, 0.1 mM Na3VO4, 0.1 mM PMSF, 2 g/mL prepulse to 80 mV or 20 mV (currents were normalized to aprotinin, 10 μg/mL leupeptin, 10 mM β−glycerolphospahte, the current following the –80 mV prepulse) or by the application × – 10 mM NaF] containing 1 Complete Mini inhibitor mixture of a 200-Hz train of 2-ms depolarizations from 80 mV to +20 (Roche). Cell extracts were vigorously vortexed for 15 min at 4 °C mV (in this case, currents were normalized to the current evoked and centrifuged at 18,000 × g for 30 min at 4 °C. by the first pulse of the train). Western Blotting. One hundred micrograms of total protein or 20 μg Capacitance and Amperometric Measurements. Capacitance meas- – fi of membrane protein were eletrophoresed in NuPAGE Novex 3 urements were performed using an EPC-9 patch-clamp ampli er 8% Tris-Acetate precast gels (Invitrogen) or 8% Tris-glicine gels, (HEKA Electronics) using the “sine + dc” software lock-in fi respectively, and transferred onto nitrocellulose membranes us- ampli er method implemented in PULSE software. The pipette ing the iBlot Dry Blotting System (Invitrogen). Membranes were solution was similar to that indicated for ICa records, and in some 2+ blocked for 1 h at room temperature in TTBS [100 mM Tris-HCl experiments the Ca buffering was changed to 0.1 or 5 mM (pH 7.5) 150 mM NaCl, 0.1% Tween 20] containing 5% skim milk EGTA to modify the magnitude of Ca2+ microdomains. Cells – powder (blocking solution). The appropriate primary and sec- were held at 80 mV except during membrane depolarization. ondary antibodies (see below) were incubated in blocking solution The assumed reversal potential was 0 mV, and the sinusoid had overnight at 4 °C or 1 h at room temperature, respectively. an amplitude of 25 mV and a frequency of 1 kHz. The stim- Membranes were subjected to chemiluminiscence analysis using fi ulation protocol was a train of ve-step depolarizations to +20 SuperSignal West Pico Chemiluminiscent Substrate (Pierce Bio- mV for 200 ms delivered at 20 Hz, as reported previously (3). technology) and detected on Curix RP2 Plus films (Agfa). Primary Single-pulse current data were leak subtracted by hyperpo- antibodies (concentration, source, and supplier) include anti- larizing sweeps.

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