Generation of NALCN mutants, cell culture, transfection, and recording

To construct various point mutants for NALCN, the pCMV2-NALCN expression vector was used as the template using the QuikChange Site-Directed Mutagenesis Kit (Agilen) as previously described¹⁹. The primers used are listed in Supplementary Table 1.

HEK293T cells (ATCC, USA, Virginia) were cultured as previously described¹⁴. Cells were maintained in Dulbecco’s modification of Eagle’s medium (Invitrogen, USA, California) supplemented with 10% fetal bovine serum. Lentiviruses of shRNA control and PRMT7 (shPRMT7) were generated with a modified lentiviral vector derived from pLKO.1 (Sigma-Aldrich, St. Louis, MO) in HEK293T cells using the helper plasmids pCMV-VSVG and pCMV delta 8.2 using Lipofectamine 2000 reagents (Invitrogen, USA, California). To generate stable cell lines, lentivirus particles were mixed in medium in the presence of 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO) for 2 days and then selected in 2 μg/ml puromycin-containing medium.

For NALCN recording, NALCN, UNC80, a constitutively active Src kinase (Src529, bearing a Y529F mutation), and CaSR cDNA were transiently cotransfected using Lipofectamine 2000 reagents as previously described²⁰. The NALCN and CaSR constructs were constructed with a vector based on pTracer-CMV2 (Invitrogen) modified to express eGFP (for NALCN) or mCherry RFP (for CaSR) under separate promoters. To obtain a heterologous KCNQ2/3 configuration, human KCNQ2 and KCNQ3 subunits with GFP plasmid were cotransfected in 293T cells and were recorded within 48–72 h after transfection as previously described²¹. The NALCN and KCNQ currents from HEK293T cells were measured with the whole-cell patch clamp technique. Voltage clamp was performed using an EPC-10 amplifier (HEKA Instruments, Germany, Lambrecht/Pfalz) at a sampling rate of 10 kHz filtered at 1 kHz. Data were acquired using an IBM-compatible computer running Patchmaster software (HEKA Instruments, Germany, Lambrecht/Pfalz). The patch pipettes were pulled from borosilicate capillaries (HilgenbergGmbH, Germany, Malsfeld) using a Narishige puller (PC-10, Narishige, Japan, Tokyo). The patch pipettes had a resistance of 2–3 MW when filled with the pipette solution containing (in mM) 140 KMeSO4, 20 KCl, 20 HEPES, 0.5 Na-GTP, 5 Mg-ATP, 4 vitamin C, and 10 1,2-bis (2aminophenoxy) ethane N,N,N_,N_-tetraacetic acid (BAPTA), pH 7.4 adjusted with KOH. The normal external solution was as follows (in mM): 143 NaCl, 5.4 KCl, 5 HEPES, 0.5 NaH2PO4, 11.1 glucose, 0.5 MgCl₂, and 1.8 CaCl₂, pH 7.4 adjusted with NaOH. Pipette capacitance was compensated after formation of a gigaohm seal. Access resistance was typically 2.8–3.2 MΩ. The perfusion system was a homemade 100-ml perfusion chamber through which the solution flowed continuously at 5 ml/min. The currents from HEK293T cells were studied by holding the cell at 60 mV, and 1-s steps from 70 to 40 mV in 10-mV increments were applied, followed by 1-s pulses to 60 mV. All recordings were carried out at room temperature (RT). Currents were analyzed and fitted using Patch master (HEKA Instruments, Germany, Lambrecht/ Pfalz) and Origin (ver. 6.0, Microcal, USA, Massachusetts) software. All values are given as the mean ± standard error. The I/V relationship was obtained by plotting the outward current at the end of a 1-s test pulse as a function of the test potential.