Expression vector
Wild-type CACNA1G (short isoform; BC110995.1, NM_198382.2) in the pCMV-SPORT6 plasmid (pCMV-SPORT6-CACNA1G) was purchased from Dharmacon (Lafayette, CO, USA). The mutation c.5075G>A, corresponding to c.5144G>A in the longest isoform (NM_018896.4), was introduced by site-directed mutagenesis using QuikChange Lightning (Agilent Technologies, Santa Clara, CA, USA) and verified by bidirectional sequencing. The IRES-EGFP sequence was amplified by PCR from the pIRES-EGFP plasmid and inserted after the termination codon in the cDNA sequence in pCMV-SPORT6-CACNA1G (pCMV-SPORT6-CACNA1G-IG) using an In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan).
Cell culture, transformation, and immunofluorescence staining
HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37 °C in an incubator with 5% CO2. Cells for whole-cell patch clamping were grown in glass-bottom plates (μ-Dish 35 mm low; Ibidi, Martinsried, Germany) for 24 h following transfection with pCMV-SPORT6-CACNA1G-IG using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA, USA).
Electrophysiology
Whole-cell recordings were obtained from GFP-expressing HEK293T cells using an upright microscope (BX51WI; Olympus, Tokyo, Japan) equipped with an IR-CCD camera system (IR-1000; DAGE-MTI, Michigan, IN, USA) at room temperature. To confirm the reproducibility of our previous study results [1], we made whole-cell recordings from cells (approximately 20% of cells used in the experiment) without blinding. For the remaining experiments with added reagent, whole-cell recordings were performed with blinding. The intracellular solution was composed of 110 mM CsCl, 20 mM TEA-Cl, 5 mM EGTA, 10 mM HEPES, 4 mM MgCl2, 4 mM 2Na-ATP, and 0.4 mM 2Na-GTP (pH 7.3, adjusted with CsOH). The pipette access resistance was approximately 2–3 MΩ. To separate the Ca2+ current, HEK293T cells were bathed with an extracellular solution containing 4-AP and a high concentration of TEA-Cl, which are broad-spectrum and non-selective potassium channel blockers. The composition of the extracellular solution (control) used for voltage-dependent Ca2+ current recording was 10 mM NaCl, 105 mM TEA-Cl, 10 mM 4-AP, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 20 mM glucose; this solution was bubbled with 95% O2 and 5% CO2. Ionic currents were recorded with an EPC-10 (HEKA Elektronik, Lambrecht, Germany). T-type calcium channel currents were activated by stepwise depolarization after hyperpolarization to − 100 mV (300 ms). The signals were filtered at 3 kHz and digitized at 20 kHz. Online data acquisition and offline data analysis were performed using the PATCHMASTER software (HEKA Elektronik). The relative conductance and steady-state inactivation potentials were fitted by the following Boltzmann equations:
$$\frac{G}{{G}_{max}}=\frac{1}{1+exp\left(\left({V}_{half}-{V}_{m}\right)/k\right)}$$
$$\frac{I}{{I}_{max}}=\frac{1}{1+exp\left(\left({V}_{m}-{V}_{half}\right)/k\right)}$$
where G and I are the conductance and the current at an individual membrane potential, respectively, and Gmax and Imax are the maximum conductance and the peak current, respectively. Vhalf, k, and Vm are the half-conductance potential, slope factor, and membrane potential, respectively. The reversal potential of the CaV3.1 current was estimated from the fitted lines of the peak amplitudes recorded at − 10, 0, + 10, + 20, and + 30 mV.
Reagent addition
The effect of zonisamide sodium salt (Sigma-Aldrich, St. Louis, MO, USA) or efonidipine hydrochloride (Cosmo Bio, Tokyo, Japan) against the electrophysiological properties of mutant CaV3.1 was examined by bathing the extracellular solution containing the reagent. The concentrations of ZNS were 0, 10, and 50 µM. This range of concentrations approximated the blood concentration of ZNS following administration of our patient, which was based on a report that the blood concentration and brain concentration are approximately equivalent [12]. The concentration of efonidipine was 5 µM, which was based on the plasma concentration in healthy adults orally administered the same amount of efonidipine as our patients and as described on the product sheet. To avoid the run-down and negative influence of insufficient washout of calcium channel blockers when recording the next cell, we obtained whole-cell recordings from cells without calcium channel blockers. Following the control experiment, we conducted whole-cell recordings from cells with calcium channel blockers. Once the reagent was applied, the dish was discarded, and the next experiment was conducted using a new dish. There was no significant difference in cell capacitance between groups.
Statistical analyses
Multiple linear regression analysis was used to study the linear relationship between a dependent variable (Ca2+ current density, the half-conductance potential, or the slope factor) and two independent variables (the presence of mutation and the concentration of the reagent [ZNS or efonidipine]). The analyses were performed for the whole-cell recordings experiments using R version 3.6.0. Numerical data are indicated as the mean ± standard deviation (SD) or as the mean ± standard error of the mean (S.E.M). The level of significance was set at p < 0.05.