Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca_v2.1 (P/Q-Type) Calcium Channels

cDNA constructs.

The cDNAs for Ca²⁺ channel α₁ subunits used in the study include human long-isoform α_1A (Ca_V2.1; AF004884; kindly provided by Dr. Joanna Jen, UCLA; Jen et al., 2001), human short-isoform α_1A (Ca_V2.1-Short; AF004883; kindly provided by Dr. Jörg Striessnig, University of Innsbruck; Wappl et al., 2002), rat α_1C (Ca_V1.2; M67515; kindly provided by Dr. Gerald Obermair, Medical University of Innsbruck; Obermair et al., 2004), and bovine α_1B (Ca_V2.2; AF173882; kindly provided by Dr. Aaron Fox, University of Chicago, and Dr. Chien-Yuan Pan, National Taiwan University; Cahill et al., 2000). Myc-tagged (C terminus) Ca_V2.1 constructs (Ca_V2.1-6myc) was created by subcloning Ca_V2.1 cDNA into a modified pcDNA3 vector (Invitrogen) with six repeats of Myc sequences right before the stop codon. The generation of GFP-tagged (N terminus) Ca_V2.1 was described previously (Jeng et al., 2008). cDNAs for human RNF128 and RNF138, as well as rat RNF138, were subcloned into the pcDNA3-Myc vector (Invitrogen). Other cDNAs include human Ca²⁺ channel auxiliary subunits α₂δ and β_4a (NM_000722 and U95020; from Dr. Jen), bovine β_1b and β_2a (AF174415 and AF174417; from Dr. Fox), HA-tagged human wild-type and lysine-less ubiquitin (HA-Ub-WT and HA-Ub-K0; kindly provided by Dr. Chihiro Sasakawa, University of Tokyo). Where indicated, additional tagged or mutation constructs were created by introducing specific sequences into appropriate regions within the cDNAs using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), followed by DNA sequence verification.

Yeast two-hybrid screening.

The distal C-terminal region of human long-isoform α_1A (Ca_V2.1-C-ter; aa 2204–2510) was used as a bait to screen a rat brain cDNA library (OriGene) by using the DupLEX-A yeast two-hybrid system (OriGene). The cDNA for the Ca_V2.1-C-ter fragment was amplified by PCR and fused in-frame to the coding sequence for the DNA-binding protein LexA, which was in turn subcloned into the yeast expression plasmid pGilda and transformed (using the lithium acetate method) into the yeast strain EGY48 that contains the reporter gene LEU2 downstream of the LexA operator. To make certain that the LexA-Ca_V2.1-C-ter fusion protein was efficiently expressed in EGY48, transformed EGY48 yeast colonies were also cultured at 30°C in 4 ml of YPD rich medium harvested right before the colony density reached the OD₆₀₀ value of 0.6, lysed in 20 μl of B-PER reagent (Pierce), and eventually subjected to immunoblotting analyses (Fig. 1A). The LexA-Ca_V2.1-C-ter-expressing EGY48 was then sequentially transformed with the reporter plasmid pSH18–34 (containing the LexA operator-lacZ fusion gene) and an activation-domain-fused cDNA library subcloned in the plasmid pJG4–5. After incubating at 30°C for 2–7 d, transformed yeast colonies growing on leucine dropout plates were scored positive for interacting proteins. Positive yeast colonies were further selected by β-galactosidase assay. Plasmid DNA extracted from yeast colonies was applied to transform the E. coli strain DH5α. Candidate cDNA clones were screened by PCR with pJG4–5-specific primers, followed by online (BLAST) and in-house sequence analyses. Of >490 candidate clones obtained with the bait, we identified 67 independent prey clones that were tested positive for Ca_V2.1 interactions. These 67 independent prey clones correspond to the partial or full amino acid sequences of ∼30 distinct proteins.

Glutathione S-transferase (GST) pull-down assays.

GST fusion proteins were produced and purified following the manufacturer's instructions (Stratagene). In brief, the cDNA fragments for human long-isoform α_1A N-terminal region (Ca_V2.1-N-ter; aa 1–99), transmembrane domains I and II linker (Ca_V2.1-I-II; aa 361–487), transmembrane domains II and III linker (Ca_V2.1-II-III; aa 715–1245), transmembrane domains III and IV linker (Ca_V2.1-III-IV; aa 1512–1567), proximal C-terminal region (Ca_V2.1-pC-ter; aa 1816–2203), and distal C-terminal region (Ca_V2.1-C-ter; aa 2204–2510) were subcloned into the pGEX vector (GE Healthcare) and expressed in the E. coli strain BL21. Bacterial cultures were grown at 30°C, induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside, and then harvested by centrifugation at 8000 × g for 10 min at 4°C. Cell pellets were resuspended in B-PER reagent (Pierce) containing 1 mm phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor mixture (Roche Applied Science). The lysates were clarified by centrifugation at 15,000 × g for 15 min, and glutathione-agarose beads (Sigma-Aldrich) were used to bind the GST fusion proteins from the supernatant. GST protein-coated beads (4–8 μg) were incubated with precleared cell lysates at 4°C overnight. The bead–protein complexes were then washed with buffer A containing the following (in mm): 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO₃, 20 Tris-HCl, pH 7.5, 1 PMSF, 1 Na₃VO₄, 1 NaF, and 1 β-glycerophosphate with and without 1% Triton X-100 and the proteins were eluted by boiling for 5 min in the Laemmli sample buffer.

Cell culture and DNA transfection.

Human embryonic kidney (HEK) 293T cells (RRID:CVCL_0063) or neuroblastoma NT2 cells (RRID:CVCL_0034; kindly provided by Dr. Thai-Yen Ling, National Taiwan University) were grown in DMEM supplemented with 2 mm glutamine, 10% heat-inactivated fetal bovine serum (Hyclone), 100 units/ml penicillin, and 50 μg/ml streptomycin and maintained at 37°C in a humidified incubator with 95% air and 5% CO₂. Transient transfection was performed by using the Lipofectamine 2000 (LF2000) reagent (Invitrogen). Briefly, cells were plated onto six- or 12-well plates (for biochemical experiments) or poly-d-lysine-coated coverslips in 24-well plates (for electrophysiological recordings) 24 h before transfection. Various expression constructs were incubated with LF2000 reagent for 20 min at room temperature and DNA-Lipofectamine diluted in Opti-MEM (Invitrogen) was applied to culture wells. After 6 h of incubation at 37°C, the medium was changed and the culture cells were maintained in a humidified 5% CO₂ incubator at 37°C for 24–48 h before being used for biochemical or electrophysiological experiments. For immunoprecipitation and ubiquitination experiments, transfected HEK293T cells were incubated in the presence of 10 μm MG132 (Sigma-Aldrich) for 24 h.

Immunoblotting.

Transfected cells were washed twice with ice-cold PBS containing the following (in mm): 136 NaCl, 2.5 KCl, 1.5 KH₂PO_4, and 6.5 Na₂HPO₄, pH 7.4, centrifuged, and resuspended in a lysis buffer containing the following (in mm): 150 NaCl, 5 EDTA, and 50 Tris-HCl, pH 7.6, 1% Triton X-100 containing a protease inhibitor mixture. After adding the Laemmli sample buffer to the lysates, samples were sonicated on ice (3 times for 5 s each) and heated at 70°C for 5 min. Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca_V2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca_V2.1 long-isoform], rabbit anti-human Ca_V2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca_V2.1], rabbit anti-rat Ca_V2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies. Blots were then exposed to horseradish-peroxidase-conjugated goat anti-mouse IgG (1:5000; Jackson ImmunoResearch catalog #115-035-003, RRID:AB_10015289), goat anti-rabbit IgG (1:5000; Jackson ImmunoResearch catalog #111-035-003, RRID:AB_2313567), or goat anti-rat IgG (1:5000; Santa Cruz Biotechnology catalog #sc-2032, RRID:AB_631755) and revealed by an enhanced chemiluminescence detection system (Thermo Scientific). Results shown are representative of at least three independent experiments. Densitometric scans of immunoblots were quantified with ImageJ (RRID:SCR_003070).

Immunoprecipitation.

Cells were solubilized in ice-cold lysis buffer containing the protease inhibitor mixture. Insolubilized materials were removed by centrifugation. Solubilized HEK293T cell lysates or brain homogenates were precleared with protein A/G Sepharose beads (GE Healthcare Biosciences) for 1 h at 4°C and then incubated for 16 h at 4°C with protein A/G Sepharose beads precoated with appropriate antibodies. Beads were gently spun down and washed twice in a wash buffer containing the following (in mm): 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO₃, and 20 Tris-HCl, pH 7.5, supplemented with 0.1% Triton X-100 and then twice with the wash buffer. The immune complexes were eluted from the beads by heating at 70°C for 5 min in the Laemmli sample buffer.

Rat brain lysates and subcellular fractionation.

All animal procedures are in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003) and approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University.

Forebrains from adult Sprague Dawley rats (RRID:RGD_5508397) were homogenized with a motor driven glass-Teflon homogenizer in ice-cold dissociation buffer containing the following (in mm): 320 sucrose, 1 MgCl_2, 0.5 CaCl_2, 1 NaHCO_3, 1 PMSF, and 1 mg/L leupeptin) and the cell debris was removed by centrifugation at 1400 × g for 10 min. The supernatant was saved and the pellet was resuspended by homogenization in ice-cold dissociation buffer and pelleted again. The remaining pellet was discarded and the combined supernatants were pelleted (13,800 × g for 10 min) again. The final pellet was resuspended in buffer A supplemented with 1% Triton X-100 and protease inhibitor mixture.

For subcellular fractionation, rat forebrains were homogenized in buffer H1 containing the following (in mm): 320 sucrose, 1 NaHCO₃, 0.5 CaCl₂, and 0.1 PMSF, along with protease inhibitor mixture, and centrifuged at 1400 × g to remove nuclei and other large debris (P1). The S1 fraction was subject to centrifugation at 13,800 × g to obtain the crude synaptosome fraction (P2). The pellet was resuspended in buffer H2 containing the following (in mm): 0.32 m sucrose and 1 mm NaHCO₃, and layered onto the top of the discontinuous sucrose density gradient by using 0.85, 1.0, and 1.2 m sucrose layers. The gradient was centrifuged at 65,000 × g for 2 h in a Beckman Instruments SW-28 rotor and the synaptosomal fraction (SPM) was recovered from the 1.0–1.2 m sucrose interface. The synaptosomal fraction was extracted in ice-cold 0.5% Triton X-100/50 mm Tris-HCl, pH 7.9, for 15 min and centrifuged at 32,000 × g for 45 min to obtain the PSD I pellet. The pellet was resuspended and further extracted a second time with 0.5% Triton X-100/50 mm Tris-HCl, pH 7.9, followed by centrifugation at 200,000 × g for 45 min to obtain the PSD II pellet. Protein concentration was determined by the BCA protein assay kit (Thermo Scientific). For immunoblotting, 40 μg (H, S1, P2, and SPM) or 20 μg (SPM, PSD I, and PSD II) of proteins were used.

Cortical neuron cultures.

Dissociated cortical cultures were prepared using a previously described protocol (Banker and Goslin, 1998) with a minor modification. In brief, the forebrains of embryonic day 18 embryos isolated from pregnant rats were removed and placed in Hank's balanced salt solution containing 10 mm HEPES, pH 7.4, and 1 mm sodium pyruvate. The cortex was dissected out and dissociated by incubation with 0.25% trypsin solution. The dissociated cells were plated on coverslips at a density of ∼160 and 800 cells/mm² for immunofluorescence and immunoblotting, respectively. Coverslips were precoated with poly-d-lysine (1 mg/ml; Sigma-Aldrich) and laminin (15 μg/ml; Sigma-Aldrich). Cultures were maintained in Neurobasal medium supplemented with B27 (2%) and Glutamax I (0.5 mm; Invitrogen).

Immunofluorescence.

Coverslips containing cortical neurons were rinsed in PBS and then fixed with methanol (at 4°C) or 4% paraformaldehyde (at room temperature) in PBS for 20 min. Cells were then permeabilized and blocked with a blocking buffer [5% normal goat serum in 20 mm phosphate buffer, pH 7.4, 0.1% (v/v) Triton X-100, and 0.45 m NaCl] for 60 min at 4°C. Appropriate dilutions of primary antibodies were applied in the blocking buffer overnight at 4°C. Immunoreactivities were visualized with goat-anti-mouse antibodies conjugated to Alexa Fluor 568 (1:200; Invitrogen catalog #A11004, RRID:AB_141371) or with goat anti-rabbit antibodies conjugated to Alexa Fluor 488 (1:200; Invitrogen catalog #A11008, RRID:AB_143165). The fluorescence images were viewed and acquired with a Leica TCS SP5 laser-scanning confocal microscope. To quantify the number of protein clusters per fixed length of neurites in cultured cortical neurons, built-in “set scale” and “freehand tool” functions of ImageJ were used to trace multiple 100 μm neurite segments, followed by counting the Ca_V2.1/RNF138/synaptophysin/PSD-95 puncta within each 100 μm neurite segment. Colocalization of Ca_V2.1/RNF138 (green punctate pixels) with synaptophysin/PSD-95 (red punctate pixels) puncta was expressed as the fraction of puncta identified as both green and red punctate pixels within each 100 μm neurite segment.

RNA interference.

Lentivirus-based shRNA constructs (subcloned into the pLKO.1 vector) targeting specific human RNF138 (RNF138-1: 5′-CCTAGCCAGATTACCAGAAAT-3′; RNF138-2: 5′-GCTAGATGAAGAAACCCAATA-3′) and Ca_V2.1 (5′-CCCTCAGGTTATTGCGTATTT-3′) sequences were purchased from the National RNAi Core Facility, Taiwan. The shRNA for GFP (5′-GACCACCCTGACCTACGGCGT-3′) was used as a control. Recombinant lentivirus was generated by transfecting HEK293T cells with the packaging plasmid pCMV-ΔR8.91, the envelope plasmid pMD.G, and shRNA expressing constructs. The virus-containing supernatant was harvested and concentrated by ultracentrifugation to yield the viral stock, which in turn was supplemented with 8 μg/ml polybrene for infection of HEK293T, NT2, or cortical cells. The infected cells were selected by 5 μg/ml puromycin and subsequently transfected with the cDNA for Ca_V2.1. For suppressing endogenous Ca_V2.1/RNF138 in cultured cortical neurons, 9 d in vitro (DIV9) neurons were infected with shRNA. After puromycin selection, infected DIV12 neurons were subject to immunoblotting analyses.

RT-PCR.

RNA was extracted from HEK293T cells or cortical neurons with TRIzol (Sigma-Aldrich), followed by phenol/chloroform separation. To eliminate potential plasmid DNA contamination in the RNA prepared from HEK293T cells transfected with Ca_V2.1, we performed DNase I (Promega) treatment as a control experiment. RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Scientific). The reaction mixture was incubated at 25°C and 37°C for 10 min and 120 min, respectively, followed by heating to 85°C for 5 min. PCR amplification with Taq polymerase (Bioman) was performed as follows: 95°C for 5 min; nonsaturating 20 (HEK293T cells) or 25 (cortical neurons) cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. The specific primers used for PCRs are as follows: HEK293T cells, Ca_V2.1 forward (5′-TGGAATCAGCGTGTTACGAG-3′) and reverse (5′-GCGGACAGAGGACTCACTTC-3′); cortical neurons, Ca_V2.1 forward (5′-TGGAATCAGCGTGTTACGAG-3′), and reverse (5′-GCAGACAGGGGACTCACTTC-3′); and GAPDH forward (5′-CAAGGTCATCCATGACAACTTTG-3′), and reverse (5′-GTCCACCACCCTGTTGCTGTAG-3′). The predicted sizes of the amplified PCR fragment for Ca_V2.1 and GAPPDH are ∼500 and 496 bp, respectively. RT-PCR products were resolved by electrophoresis in 1.7% agarose gels, followed by quantification with ImageJ.

Cycloheximide chase.

At 24 h after transfection, cells were subjected to treatment with the protein synthesis inhibitor cycloheximide (100 μg/ml; Sigma-Aldrich) for 0–6 h, followed by immunoblotting. Quantitative analyses of protein degradation time course under various coexpression conditions were implemented by standardizing Ca_V2.1 protein densities in response to different CHX treatment durations as the ratio of Ca_V2.1 signal to the cognate tubulin signal, followed by normalization to the corresponding vector control at 0 h. The logarithmic value of normalized attenuation of Ca_V2.1 protein density was in turn plotted against a linear scale of CHX treatment duration (the semilogarithmic plot). The Ca_V2.1 protein half-life value was then derived from single/double linear-regression analysis of the semilogarithmic plot. For a given experimental condition with multiple repeats, an individual Ca_V2.1 protein half-life value was determined from each single trial, followed by data pooling and statistical analyses.

Protein ubiquitination analyses.

Cells were solubilized in lysis buffer supplemented with 2.5 mg/ml N-ethylmaleimide, followed by immunoprecipitation with the anti-Myc antibody as described above. To facilitate the visualization of ubiquitination signals associated with proteins of high molecular weight, a 7% SDS-PAGE protocol involving a gel running time over 2 h was used.

Biotinylation of cell surface proteins.

Transfected cells were washed extensively with d-PBS (Sigma-Aldrich) supplemented with 0.5 mm CaCl₂, 2 mm MgCl₂, followed by incubation in 1 mg/ml sulfo-NHS-LC-biotin (Thermo Scientific) in d-PBS at 4°C for 1 h with gentle rocking. Biotinylation was terminated by removing the biotin reagents and rinsing three times with 100 mm glycine in PBS, followed by once in TBS buffer containing the following (in mm): 20 Tris-HCl and 150 NaCl, pH 7.4. Cells were solubilized in a lysis buffer containing the following (in mm): 150 NaCl, 50 Tris-HCl, 1% Triton X-100, 5 EDTA, 1 PMSF, pH 7.6 supplemented with the protease inhibitor mixture. Insolubilized materials were removed by centrifugation. Solubilized cell lysates were incubated overnight at 4°C with streptavidin-agarose beads (Thermo Scientific). Beads were washed once in the lysis buffer, followed by twice in a high-salt buffer containing the following (in mm): 500 NaCl, 5 EDTA, 50 Tris-HCl, pH 7.6, 0.1% Triton X-100 and once in a low-salt buffer containing the following (in mm): 2 EDTA, 10 Tris-HCl, pH 7.6, 0.1% Triton X-100. The biotin–streptavidin complexes were eluted from the beads by heating at 70°C for 5 min in the Laemmli sample buffer.

Electrophysiological recordings.

Both Xenopus oocytes and HEK293T cells were used for functional studies of human Ca_V2.1 currents. For in vitro transcription, cDNAs were linearized with appropriated restriction enzymes as described previously (Jeng et al., 2006). Capped cRNAs were transcribed from the linearized cDNA template with the mMessage mMachine T7 kit (Ambion). For cRNA injection, adult female Xenopus laevis (African Xenopus Facility) were anesthetized by immersion in ice water containing Tricaine (1.5 g/L). Ovarian follicles were removed from Xenopus frogs, cut into small pieces, and incubated in the ND96 solution containing the following (in mm): 96 NaCl, 2 KCl, 1.8 MgCl₂, 1.8 CaCl₂, and 5 HEPES, pH 7.2. To remove the follicular membrane, Xenopus oocytes were incubated in the Ca²⁺-free ND96 solution containing collagenase (2 mg/ml) on an orbital shaker (∼200 rpm) for ∼60–90 min at room temperature. After several washes with collagenase-free, Ca²⁺-free ND96, oocytes were transferred to ND96. Stage V–VI Xenopus oocytes were then selected for cRNA injection. Unless stated otherwise, cRNAs (and cDNAs) for α_1A, α₂δ, and β₄ were mixed in a molar ratio of 1:2:2 and the final injection amounts (total volume: 41.4 nl) for α_1A, α₂, and β₄ cRNAs were ∼3.8 ng, ∼3.8 ng, and ∼1.9 ng per oocyte, respectively. Injected oocytes were stored at 16°C in ND96. 2–3 d after cRNA injection, Xenopus oocytes were transferred into a recording bath (∼200 μl) containing the following (in mm): 40 Ba(OH)₂, 50 NaOH, 2 CsOH, 5 HEPES, and 0.4 niflumic acid, pH 7.4 with methanesulfonic acid. An agarose bridge was used to connect the bath solution to a ground chamber (containing 3 m KCl), into which two ground electrodes were inserted. Borosilicate electrodes (0.1–1 MΩ) used in voltage recording and current injection were filled with 3 m KCl. Ba²⁺ currents through Ca_V2.1 channels were acquired using the conventional two-electrode voltage-clamp technique with an OC-725C oocyte clamp (Warner Instruments).

Conventional whole-cell patch-clamp technique was performed in HEK293T cells. Transfected cells were identified by GFP fluorescence (the cDNAs for Ca_V2.1 and pEGFP were cotransfected in the molar ratio 1:0.05) with an inverted fluorescence microscope (Leica DM IRB). Recording electrodes (2–4 MΩ) were pulled by a PP-830 puller (Narashige). The pipette solution contained the following (in mm): 145 CsCl, 3 MgCl₂, 5 EGTA, 10 HEPES, 2 Mg-ATP, and 0.4 Na-GTP, pH 7.3. The bath solution contained the following (in mm): 10 BaCl₂, 135 TEA · Cl, 4 KCl, 1 MgCl₂, and 10 HEPES, pH 7.2. Data were acquired and digitized with Axopatch 200B and Digidata 1440A, respectively, via pCLAMP 10.2 (Molecular Devices, RRID:SCR_011323). Cell capacitances were measured using the built-in functions of pCLAMP 10.2 and were compensated electronically with Axopatch 200B. The holding potential was set at −90 mV and passive membrane properties were compensated using the −P/4 leak subtraction method provided by the pCLAMP software. Data were sampled at 10 kHz and filtered at 1 kHz. All recordings were performed at room temperature (20–22°C).

To avoid the potential biases imposed by variations in Ca_V2.1 WT channel expression among different oocytes, we adopted a previously reported data normalization procedure (Jeng et al., 2006). Briefly, for the same batch of oocytes on a given day of experimentation, the average value of the peak Ba²⁺ current amplitudes of Ca_V2.1 WT channels under each coexpression condition was normalized to that of Ca_V2.1 WT vector control. Normalized data from different batches of Xenopus oocytes on different dates were later pooled together for comprehensive analysis. A similar Ba²⁺ current density normalization and data pooling strategy was also applied to the HEK293T cell system

Statistical analyses.

All values are presented as mean ± SEM. The significance of the difference between two means was tested using Student's t test, whereas means from multiple groups were compared using one-way ANOVA analysis. All statistical analyses were performed with Origin version 7.0 (Microcal Software, RRID:SCR_002815).