The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells

Journal Pre-proof The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells Yingying Ye, Mohammad Barghouth, Cheng Luan, Abdulla Kazim, Yuedan Zhou, Lena Eliasson, Enming Zhang, Ola Hansson, Thomas Thevenin, Erik Renström PII:

S0303-7207(19)30375-2

DOI:

https://doi.org/10.1016/j.mce.2019.110673

Reference:

MCE 110673

To appear in:

Molecular and Cellular Endocrinology

Received Date: 26 March 2019 Revised Date:

19 November 2019

Accepted Date: 30 November 2019

Please cite this article as: Ye, Y., Barghouth, M., Luan, C., Kazim, A., Zhou, Y., Eliasson, L., Zhang, E., Hansson, O., Thevenin, T., Renström, E., The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/j.mce.2019.110673. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

The TCF7L2-dependent High-Voltage Activated Calcium Channel Subunit α2δ-1 Controls Calcium Signaling in Rodent Pancreatic Beta-cells Yingying Ye1, Mohammad Barghouth1, Cheng Luan1, Abdulla Kazim1, Yuedan Zhou2, Lena Eliasson1, Enming Zhang1, Ola Hansson2, Thomas Thevenin1* and Erik Renström1*

1

Lund University, Department of Clinical Sciences, Islet Pathophysiology Group

2

Lund University, Department of Clinical Sciences, Diabetes and Endocrinology Group

*Correspondence to Thomas Thevenin or Erik Renström Lund University, Department of Clinical Sciences, Islet Pathophysiology, Clinical Research Centre, Att: T.T. or E.R. Box 50332, SE-205 13 Malmö, Sweden Phone +46040391157 Fax +46040391210 e-mail: [email protected]

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Abstract The transcription factor TCF7L2 remains the most important diabetes gene identified to date and genetic risk carriers exhibit lower insulin secretion. We show that Tcf7l2 regulates the auxiliary subunit of voltage-gated Ca2+ channels, Cacna2d1 gene / α2δ-1 protein levels. Furthermore, suppression of α2δ-1 decreased voltage-gated Ca2+ currents and high glucose/depolarization-evoked Ca2+ signaling which mimicked the effect of silencing of Tcf7l2. This appears to be the result of impaired voltage-gated Ca2+ channel trafficking to the plasma membrane, as Cav1.2 channels accumulated in the recycling endosomes after α2δ-1 suppression, in clonal as well as primary rodent beta-cells. This impaired the capacity for glucose-induced insulin secretion in Cacna2d1-silenced cells. Overexpression of α2δ-1 increased high-glucose/K+-stimulated insulin secretion. Furthermore, overexpression of α2δ-1 in Tcf7l2-silenced cells rescued the Tcf7l2-dependent impairment of Ca2+ signaling, but not the reduced insulin secretion. Taken together, these data clarify the connection between Tcf7l2, α2δ-1 in Ca2+-dependent insulin secretion. Keywords Tcf7l2; α2δ-1; Type 2 diabetes Abbreviations T2D

Type 2 diabetes

Tcf7l2 T-cell factor 7-like 2 VGCC Voltage-gated calcium channels [Ca2+]i

Free intracellular calcium

PM

Plasma membrane

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1. Introduction Type 2 diabetes develops when the demand for insulin exceeds what the pancreatic beta cells can deliver. Glucose-stimulated insulin secretion occurs by Ca2+-dependent exocytosis and in this respect, Ca2+ entering via voltage-gated calcium channels (VGCCs) plays a particularly important role (Rorsman, Eliasson et al. 2000). Pancreatic β-cells are equipped with at least six types of calcium channels, including Cav1.2, Cav1.3, Cav2.1, Cav2.2, Cav2.3 and Cav3.1 (Yang and Berggren 2006), of which L-type calcium channels (Cav1.2 and Cav1.3) conduct ~50% of the whole Ca2+ currents (Schulla, Renstrom et al. 2003). Nitert et al. reported that the mRNA level of Cav1.2 exceeded that of Cav1.3 and Cav2.3 two-fold in INS-1 832/13 cells, and suggested that Cav1.2 rather than Cav1.3 is critical to glucose-stimulated insulin secretion in INS-1 832/13 cells (Nitert, Nagorny et al. 2008). Rorsman et al. concluded that Cav1.2 is the principal L-type Ca2+-channels subtype in mouse β-cells (Rorsman, Braun et al. 2012). VGCCs are multi-subunit proteins consisting of the pore-forming α1 subunit, which exists in ten different isoforms in mammalian genomes and is the major VGCC subunit determining the main physiological and pharmacological properties. In addition, a number of auxiliary subunits exist in the genome, each in different isoforms, which attach to the α1 pore subunit and modulate its functions. Of the auxiliary subunits, the function of the γ subunits remains largely unknown, whereas the α2δ and β subunits have been suggested to control VGCC trafficking to plasma membrane (PM), but also to influence certain of the channels’ biophysical properties (Dolphin 2013). Accordingly, in heterologous expression systems the α2δ subunits increase the maximum current density of Cav1 and Cav2 VGCCs (Dolphin 2003). The main mechanism has been suggested that increased expression of Cav1 and Cav2 complexes at the plasma membrane coupled to a decrease in their turnover (Canti, Nieto-Rostro et al. 2005, Bernstein and Jones 2007). Interestingly, it was demonstrated in mouse that genetic ablation of α2δ-1 (the major variant in pancreatic islets) reduces Ca2+ influx via all types of functional VGCCs in the pancreatic beta cells, which resulted in reduced insulin secretion and impaired glucose tolerance, at least in male mice (Mastrolia, Flucher et al. 2017). 3

However, a detailed characterization of the cellular effects at the single beta cell level remains to be performed. TCF7L2 (T-cell factor 7-like 2, also known as TCF4) harbors the single nucleotide polymorphism (SNP) rs7903146, which remains the most significantly common genetic variation associated with human type 2-diabetes (Grant, Thorleifsson et al. 2006). This genetic variant is linked to reduced gene expression, in particular of certain splice variants (Osmark, Hansson et al. 2009), and reduced capacity for insulin secretion (Lyssenko, Lupi et al. 2007), but pathophysiological role of TCF7L2 in development of type 2-diabetes is elusive. Recently, report showed that TCF7L2 is a master regulator of insulin production and processing (Zhou, Park et al. 2014). By contrast TCF7L2 has no major influence over genes involved in the control of Ca2+ signaling and exocytosis, with one exception: Cacna2d1 gene expression is markedly reduced in Tcf7l2 silenced cells (Zhou, Park et al. 2014). However, the functional consequences in terms of protein levels and beta cell function are still missing. Therefore, we decided to extend on these previous reports by verifying the regulatory role of the diabetes gene Tcf7l2 on α2δ-1 expression, as well as its consequences for Ca2+ signaling and insulin secretion in the beta cell.

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2. Materials and Methods 2.1. Cell culture and preparation of islets, β cells Rat-derived INS-1 832/13 cells were cultured in RPMI 1640 (HyClone, USA) medium containing 11.1 mΜ D-glucose (Sigma, USA) supplemented with 10% fetal bovine serum (Sigma, USA), 11.2 mM HEPES (HyClone, USA), 100 U/ml penicillin (HyClone, USA), 100 µg/ml streptomycin (HyClone, USA), 2mM L-glutamine (HyClone, USA), 1 mM sodium pyruvate (HyClone, USA) and 0.05 mΜ 2-mercaptoethanol (Sigma, USA), at 37°C in a humidified atmosphere containing 95% air and 5% CO2. The INS-1 832/13 cells were stored at generation passage number 52, in this study, passage #54-#75 of INS-1 832/13 cells were used. Male rats /mice used in this study were between 10-20 weeks old. Primary rat/mouse pancreatic islets were isolated as described earlier (Fransson, Rosengren et al. 2006) and handpicked under a stereomicroscope. Whole islets were cultured in petri dishes (Sarstedt, USA) containing RPMI 1640 (HyClone, USA) as above but substituted with 5 mM (for rat islets)/ 10 mM (for mouse islets) Dglucose and lacking 2-mercaptoethanol. Mouse pancreatic islets were isolated by gentle collagenase digestion and were subsequently maintained in a short-term tissue culture medium as mentioned above. All the animal experiments were referred to ARRIVE guidelines (Kilkenny, Browne et al. 2012). 2.2. RNA interference INS-1 832/13 cells were seeded 24 h prior to transfection. 20 nM rat-targeted Cacna2d1 or Tcf7l2 RNA interference oligonucleotides (Thermofisher Scientific, USA) or 20 nM negative control #1 (Thermofisher Scientific, USA) were used to silence Cacna2d1 or Tcf7l2 using Dharmafect Kit (Dharmacon, USA) or Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, USA) the next day. After transfection for 48 h, the cells were collected for mRNA extraction or other measurements.

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For silencing in rat islets, freshly isolated islets were picked, counted and directly transfected using the same RNA interference oligonucleotides as above and following the protocol of Lipofectamine® RNAiMAX transfection reagent. After 72 h transfection, the islets were collected for mRNA extraction. A mouse-targeted RNA interference oligonucleotides of Cacna2d1 was used for transfection in mouse islets, the islets were dispersed after 48 h transfection and cultured in µ-Slide (chambered coverslip) with 8 wells (ibidi, Germany) for another 24 h followed by immunostaining, or seeded in 35-mm Nunc plastic petri dishes with or without treatment of GBP followed by electrophysiology. 2.3. Overexpression of α2δ-1 The α2δ-1 cDNA was produced from the plasmid rat-α2δ-1-pMT2 (Dolphin 2013) which was a gift from Annette Dolphin (Addgene plasmid # 58726), and then this cDNA was inserted into the lentivirus backboned vector with mCherry (pLenti-C-mCherry), the recombinated DNA (pLenti-α2δ1-mCherry) was confirmed by DNA sequencing. INS-1 832/13 cells were transfected with Tcf7l2 RNA interference oligonucleotides or negative control 24 h prior to virus transduction. Then 0.5 titer/cell of α2δ-1-mCherry lenti-virus was used to transduce the α2δ-1 cDNA into those siRNA transfected INS-1 832/13 cells and mCherry lenti-virus was served as control. The cells were cultured for another 48 h for later measurements. 2.4. Real-time quantitative PCR Total mRNA was extracted from the transfected cells or rat/mouse islets using RNeasy Mini kit (Qiagen, Germany) and reverse transcripted (SuperScript Ⅲ , Invitrogen, USA) following the manufacturers’ instructions. The resulting cDNA was subjected to 40 cycles of quantitative real-time PCR (RotorGene 2000, Corbett Research) in 10 µl reactions containing 2× universal PCR master Mix (TaqMan, USA) and 0.2 mM primers. Data was normalized to the expression levels of hypoxanthine guanine phosphoribosyl transferase1 (Hprt1) in each sample. Primers of Cacna2d1, Tcf7l2, Cacna1c, 6

Stxbp1, Syt14, Vamp2 and housekeeping gene Hprt1 (Thermofisher Scientific, USA) were used for amplification detection. 2.5. Immunostaining Immunostaining in transfected INS-1 832/13 cells and dispersed mouse islet β cells was done as described previously (Buda, Reinbothe et al. 2013). Cells were first washed twice with PBS (Hyclone, USA) and fixed with 3% PFA-K-PIPES (pH6.5) and 3% PFA-Na2BO4 (pH11) for 5 and 10 min respectively, followed by permeabilization with 0.1% Triton-X 100 for 30 min. The blocking solution contained 5% normal donkey serum (Jackson immunoresearch, USA) in PBS was used for 30 min. Primary antibodies against Cav1.2 (1:200, Sigma, USA), Na+/K+-ATPase (1:200, Millipore, USA), Rab11 (1:200, BD transduction lab, USA), Insulin (1:400, EuroProxima, Arnhem, the Netherlands) were diluted in blocking solution and incubated overnight at 4°C. Immunoreactivity was detected by fluorescently labeled secondary antibodies (1:400) and visualized by confocal microscopy (Carl Zeiss, Germany). Colocalization analysis was performed using a ZEN2012 software based on Pearson’s correlation coefficient analysis which recognizes the colocalized pair by comparing pixel by pixel intensity (Costes, Daelemans et al. 2004). The internalization was indicated by ratio that is defined by mean intensity of plasma membrane to mean intensity of cytosol, according to the formula: Ratio = (

×

×

)/(

×

×

) where i1, i2 and i3 represent the intensities of whole cell, cytosol plus

nulceus and nucleus respectively, and a1, a2 and a3 represent the area of whole cell, cytosol plus nucleus and nucleus respectively. The specificity of Cav1.2 antibody was validated using synthesized peptide which totally blocked the signals. 2.6. Ca2+ imaging Intracellular Ca2+ was measured as previously described (Buda, Reinbothe et al. 2013). Cells were first washed with Krebs-Ringer bicarbonate (KRB) buffer containing 116 mM NaCl, 4.7 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 20 mM NaHCO3, 16 mM HEPES, and supplemented with 5 7

mM glucose and then incubated with 1 mM Fluo-5F AM (Invitrogen, USA) at 37°C for 30 min. Stimulation was carried out using 70 mM KCl KRB buffer alone or 16.7mM glucose for 10min followed by high K+ solution. Images were acquired by confocal microscopy using 63 × oil immersion objectives. A ratio was calculated by taking the fluorescence intensity in time lapse divided by the average fluorescence intensity under pre-stimulatory conditions. The peak amplitude was normalized to basal fluorescence intensity. 2.7. Subcellular fractionation Transfected INS-1 832/13 cells were lysed and fractionated using a Qproteome Cell Compartment kit (Qiagen, Germany) according to the manufacturer’s instructions. Briefly, cells were scraped into a 15 ml conical tube and centrifuged at 500×g for 10 min at 4°C. The cell pellet was washed in ice-cold PBS twice and resuspended in ice-cold Extraction Buffer CE1 with protease inhibitor solution. The pellet was then incubated for 10 min at 4°C on an end-over-end shaker. The lysate was centrifuged at 1000×g for 10 min at 4°C and the supernatant containing cytosolic proteins was collected. The pellet was resuspended in 1 ml ice-cold Extraction Buffer CE2 with protease inhibitor solution and incubated for 30 min at 4°C on an end-over-end shaker. The suspension was centrifuged at 6000×g for 10 min at 4°C and the supernatant containing membrane proteins was collected. The fractions were then isolated by acetone precipitation overnight on ice. The pellets were centrifuged at 12,000×g, 4°C, 10 min and dissolved in urea buffer containing 8 M urea, 50 mM Tris-HCl (pH 7.4), and 100 mM NaCl. Subcellular fractions were quantified using BCA protein assay kit (Pierce, USA) and subjected to immunoblotting. Endosome extreaction was done using the Minute™ Endosome Isolation and Cell Fractionation Kit (ED-028, inventbiotech, USA) following the instruction, the resulted endosome pellet was washed with PBS+0.3M NaCl (pH 9.5) once and centrifuge at 16,000 ×g for 10 min at 4°C. The final pellet was resolved in RIPA buffer and lysed on ice for at least 30 min. 8

For immunoblotting, 50 µg of plasma membrane fractions or 15 µg of endosome fractions was loaded onto 8% or 4-15% SDS-PAGE gels, respectively. Blotting was carried out by incubation overnight at 4°C with polyclonal anti-Cav1.2 antibody (1:400), anti-α2δ-1 antibody (1:1000), anti-Na+/K+-ATPase antibody (1:1000), anti-β-actin (1:1000), anti-SOD4 antibody (1:2000), anti-Rab11 antibody (1:400) followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibodies (1:1000) for at least 1 hour. Final signal was indicated by SuperSignal West Pico Chemiluminescent Substrate (Pierce, USA) and SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, USA). 2.8. Electrophysiology Mouse islets were dispersed after 48 h transfection and seeded in the 35-mm Nunc plastic petri dishes treating with or without 5 mM gabapentin (GBP) for 24 h. Whole-cell Ca2+ currents were measured as described previously (Luan, Ye et al. 2019). The extracellular solution contained 118 mM NaCl, 20 mM tetraethylammonium chloride, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 5 mM HEPES and 5 mM Glucose. The pipette (intracellular) solution contained 125 mM Cs-glutamate, 125 mM Lglutamic acid, 10 mM CsCl, 10 mM NaCl, 1 mM MgCl2, 5 mM HEPES, 3 mM Mg-ATP, 0.1 mM cAMP and 1.5 mM Bapta (pH 7.15 with CsOH). INS-1 832/13 cells pretreated with GBP for 24 h were used for eliciting exocytosis in the standard whole-cell configuration, in which the pipette solution dialyses the cell and replaces the cytosol, the pipette solution consisted of 125 mM K-glutamate, 10 mM KCl, 10 mM NaCl, 1 mM MgCl2, 5 mM HEPES, 3 mM Mg-ATP, 10 mM EGTA and 9 mM CaCl2, 0.1 mM cAMP (pH 7.2 with KOH). The resulting free intracellular Ca2+ concentration was estimated to 1.5 µM according to the binding constants of Martell & Smith. Data was recorded on a HEKA EPC9 patch clamp amplifier with the Pulse Fit 8.64 software. The pipettes had an average resistance of ≈ 5.5 MΩ. All the experiments were performed in bath-heated perfusion system which controls output temperature to 32°C.

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2.9. Insulin secretion assays The transfected INS-1 832/13 cells with 100% confluence were washed twice with SAB buffer (pH 7.2) containing 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 25.5 mM NaHCO3, 2.6 mM CaCl2, 20 mM HEPES, 0.2% BSA, and preincubated in 2.8 mM glucose SAB buffer for 2 h at 37 °C. Insulin secretion was then induced by static incubation of cells for 1 h in buffer containing 2.8 or 16.7 mM glucose, or 10 min for 50 mM K+ stimulation. Insulin from INS-1 832/13 cells was measured with rat insulin high range ELISA kit (Mercodia, Sweden) and normalized according to protein content per well. Protein content was determined using a BCA-assay kit. 2.10. Statistical Analysis The data were presented as means ± S.E.M. Evaluation of statistical significance was done using Student’s t-test either paried or unpaired comparison or one-way / two-way ANOVA multiple comparisons for experiments.

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3. Results 3.1. Tcf7l2 controls expression of Cacna2d1/ α2δ-1 To assess the effect of Tcf7l2 on expression of Cacna2d1, INS-1 832/13 cells were first pre-treated with Tcf7l2 targeting siRNA and non-targeting siRNA for 48 h. Silencing efficiency on the mRNA level amounted 65%±1% (Fig. 1A). Tcf7l2 silencing resulted in a significant decrease in gene expression of Cacna2d1 (Fig. 1B). Similar results were observed in primary rat islets in which silencing of Tcf7l2 (Fig. 1C) also induced a significant decrease in the expression of Cacna2d1 (Fig. 1D), even though the transfection efficiency was somewhat lower than in INS-1 832/13 cells (Fig. 1C). These results corroborate our previous report using RNA sequencing to the effect that Cacna2d1 gene expression decreases after Tcf7l2 silencing (Zhou, Park et al. 2014). To test whether these effects also relay onto altered protein translation, we performed immunoblot analysis in Tcf7l2-silenced cells, which showed reduced levels of α2δ-1 protein by 60%±11% (Fig. 1E and F). 3.2. Silencing of Cacna2d1 prevents trafficking of Cav1.2 to the plasma membrane Previous reports suggest that α2δ-subunits promote α1-subunit trafficking to the plasma membrane (Khosravani and Zamponi 2006), meanwhile silencing of Tcf7l2 has no significant effect on the expression of the major α-subunits of the VGCCs (Zhou, Park et al. 2014). Therefore, we nevertheless hypothesized that α2δ-1, the target protein of Tcf7l2 may play a role in regulation of VGCC trafficking in the beta cell. In order to clarify this, we first verified in INS-1 832/13 cells that silencing of Cacna2d1 did not affect Cacna1c gene expression (Fig. 2A and B). Similar results were also observed in primary rat islets in which the silencing of Cacna2d1 did not decrease Cacna1c expression (Fig. 2C and D). These results were confirmed by immunoblotting that showed no reduction of Cav1.2 protein in Cacna2d1–silenced cells (Fig. 2E-G). To test whether α2δ-1 plays a role in the trafficking of Cav1.2 to the plasma membrane in beta cells, we used cellular fractionation. Results showed that although the total cellular amounts of Cav1.2 11

remain unchanged, the percentage of Cav1.2 at the plasma membrane decreased significantly (Fig. 2H and I) after knockdown of Cacna2d1. This strongly suggests that α2δ-1 is involved in Cav1.2 trafficking to the plasma membrane in insulin-secreting cells. Previous reports have demonstrated that Cav1.2 cycles between recycling endosomes and plasma membrane (Buda, Reinbothe et al. 2013). We therefore investigated the localization of Cav1.2 in the recycling endosome. Immunoblotting result showed that the amount of Cav1.2 in the endosome was increased significantly in Cacna2d1-silenced INS-1 832/13 cells (Fig. 2J and K). 3.3. Silencing of Cacna2d1 retains Cav1.2 in recycling endosomes To allow for analysis of Cav1.2 location in different subcellular localizations, we next performed coimmunostaining of Cav1.2 and the plasma membrane marker Na+/K+-ATPase in single INS-1 832/13 cells. The ratio of mean intensity of Cav1.2 in the plasma membrane over that in the cytosol was used to quantify plasma membrane expression of Cav1.2. This ratio was markedly reduced after Cacna2d1 silencing (from 0.63±0.07 to 0.36±0.03, Fig. 3A and B). The same experiment in mouse islet β cells was conducted, but the cells were stimulated with 20 mM glucose and recover in 2.8 mM glucose medium for 30 min before immunostaining, it showed similar outcome, Cacna2d1 knockdown decreased plasma membrane expression of Cav1.2 (from 0.49±0.03 to 0.34±0.03, Fig. 3E and Suppl. Fig. 1A). Together with the cell fractionation data above, these results clearly demonstrate that Cacna2d1 silencing affects Cav1.2 trafficking and prevents its functional plasma membrane location. We also confirmed the endosomal localization of Cav1.2, as suggested by immunoblotting, by confocal immunocytochemistry showing co-localization of Cav1.2 and the recycling endosome marker Rab11 in Cacna2d1-silenced INS-1 832/13 cells (Fig. 3C) and mouse islet β cells (Suppl. Fig. 1B). The results showed significantly increased Cav1.2 co-localization with Rab11 after Cacna2d1 knockdown (from 0.08±0.008 a.u. to 0.28±0.02 a.u., Fig 3D. from 0.017±0.012 a.u. to 0.06±0.014 a.u, Fig. 3F). 12

Tcf7l2-silencing resulted in reduction of α2δ-1, we next studied whether trafficking of Cav1.2 could be affected as well. Immunostaining data showed that by lenti-viral transduction of α2δ-1 in Tcf7l2-silenced INS-1 832/13 cells (Suppl. Fig. 3) increased expression of membrane Cav1.2, while Tcf7l2-silencing alone did not affect Cav1.2 trafficking to the membrane (Fig. 3G and H). 3.4. Silencing of Cacna2d1 affects Ca2+ influx and exocytosis The data presented so far demonstrate that Tcf7l2 controls Cacna2d1 gene and α2δ-1 protein expression, which in turn is important for regulating plasma membrane location of Cav1.2. Next, we investigated whether these maneuvers also modulate Ca2+ entry under depolarization-evoked or high glucose- stimulated conditions. To this end we either silenced Cacna2d1 by siRNA, or inhibited it by an anti-epileptic drug gabapentin (GBP) that binds to an epitope in α2δ-1 and α2δ-2. Study showed that long-term treatment (>24 h) of GBP inhibits Ca2+ currents in dorsal root ganglion (DRG) neurons, in a fashion that mimics lack of α2δ (Hendrich, Van Minh et al. 2008). As read-out we measured free intracellular Ca2+ ([Ca2+]i) stained with fluo-5F by high resolution confocal imaging. The cells were depolarized by a step increase in extracellular [K+] from 5.6 to 70 mM, resulting in a peak of intracellular [Ca2+]i. Knockdown of Cacna2d1 resulted in a significant drop in high-K+ induced [Ca2+]i peaks, from 16±1 a.u. in controls to 12±1 a.u. in Cacna2d1-silenced cells (Fig. 4A and B). GBP treatment (24 h) reduced the [Ca2+]i peaks in both control-treated cells (NC), and Cacna2d1-silenced cells (Fig. 4A and B). We also measured whole-cell Ca2+ current-voltage relations (I-Vs) in mouse islet β cells to estimate if Cacna2d1 knockdown or GBP treatment suppresses Ca2+ influx. Both silencing of Cacna2d1 and 24 h-treatment with GBP reduced whole-cell Ca2+ currents significantly (Fig. 4C). Since Tcf7l2 controls expression of Cacna2d1/α2δ-1, we predicted that silencing Tcf7l2 should also reduce depolarizationevoked [Ca2+]i peaks and this assumption was corroborated by the experimental data (Fig. 4D and E). Together, these results show that, α2δ-1 regulates voltage-gated Ca2+ influx in insulin-secreting β cells. 13

Furthermore, this regulatory step is under the control of Tcf7l2 that thereby also controls Ca2+ influx, albeit in an indirect manner. We also measured whether silencing of Cacna2d1 or Tcf7l2 had the same effect on Ca2+ signaling under high glucose stimulation which mimicked a physiological condition. As expected, we observed fewer oscilations of Ca2+ influx after high glucose stimulation in Cacna2d1 or/and Tcf7l2-silenced cells (Fig.5A-D). We next investigated whether these effects extend to also influence glucose-induced insulin secretion (GSIS). Insulin secretion in Cacna2d1 or/ and Tcf7l2-silenced or negative control-treated INS-1 832/13 cells was stimulated with 2.8 or 16.7 mM glucose for 1 h and then assayed. As expected, silencing Cacna2d1 decreased high glucose-stimulated insulin secretion (Fig. 5E). Silencing of Tcf7l2 further reduced insulin secretion compared to that in Cacna2d1-silenced cells (Fig. 5E). Double knockdown of both these genes showed most dramatic reduction of glucose-stimulated insulin secretion (Fig. 5E), which suggested that other genes might be involved in Tcf7l2 regulation for glucose-stimulated insulin secretion. Surprisingly, GBP treatment failed to affect insulin release (data not shown). To explore whether a direct stimulatory effect by GBP on the exocytotic machinery could counteract the decreased Ca2+ stimulus, we next used the standard whole-cell configuration of the patch clamp technique, to verify the GBP effect on exocytosis itself. A patch electrode solution containing a Ca2+/EGTA buffer with [Ca2+]i free ~1.5 µM was thus dialyzed intracellularly to evoke exocytosis measured as increases in cell capacitance (Suppl. Fig. 2). This experiment showed that exocytosis occurred at similar rate in INS-1 832/13 cells treated, or not, with GBP. 3.5. Overexpression of α2δ-1 partially counteracts the effect of silencing Tcf7l2 To further investigate whether rescued expression of α2δ-1 in Tcf7l2-silenced cells could compensate the defect of Tcf7l2 deletion, we overexpressed α2δ-1 using lentivirus either in control cells or Tcfl7l2silenced cells (Suppl. Fig. 3). High-glucose stimulated Ca2+ signaling was not altered by 14

overexpression of α2δ-1 in control cells (Fig. 6A and C). However, in Tcf7l2-silenced cells, overexpression of α2δ-1 increased Ca2+ responses, demonstrating successful counteraction of the suppressive effect of Tcf7l2 silencing (Fig. 6B and C). Previous RNA sequencing data has shown that Tcf7l2 depletion in INS-1 832/13 cells reduces expression of genes involved in the exocytotic machinery (Zhou, Park et al. 2014). Our data confirm this and we demonstrate that silencing of Tcf7l2 decreased mRNA expression of Syt14 (Synaptotagmin-14), Stxbp1 (syntaxin binding protein 1, also known as Munc 18-1) and Vamp2 (Vesicle-associated membrane protein 2). Interestingly, silencing of Cacna2d1 had the identical effects as depletion of Tcf7l2 on Syt14 and Stxbp1 (Fig. 6D and E) but did not reduce expression of Vamp2 (Fig. 6F). However, overexpression of α2δ-1 failed to rescue expression of Syt14, Stxbp1 and Vamp2 after Tcf7l2-silencing (Fig. 6G and I). Thereafter, we assessed insulin secretion under the same conditions as above. Overexpression of α2δ-1 in control INS-1 832/13 cells increased both high K+induced and high glucose-stimulated insulin secretion significantly (Fig. 6J and K). However, insulin secretion was not improved by overexpression of α2δ-1 in Tcf7l2-silenced cells (Fig. 6J and K), which is likely explained by the failure of the subunit to reverse the negative effect of Tcf7l2 silencing on expression of the exocytotic genes Syt14, Stxbp1 and Vamp2. The pure effect of silencing Tcf7l2 without affection of lentivirus-Cherry on high-glucose stimulated insulin secretion was done in parallel, it showed a remarkedly reduction after silencing of Tcf7l2 (Fig. 6L).

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Discussion Tissue distribution of the different α2δ subunit isoforms, as well as their subcellular expression is thoroughly studied. The α2δ-1 subunit is primarily expressed in excitable cells (Gong, Hang et al. 2001). In the present study, α2δ-1 exhibited significant expression in insulin-secreting β cells and was regulated by the transcription factor Tcf7l2 (Fig. 1). The limitation of lower efficacy of silencing in rat /mouse islets could be complemented by studies in conditional knockout mice in the future. The α2δ subunit is known to associate with Cav1.2 (Marais, Klugbauer et al. 2001), but α2δ-1 does not control gene expression or protein levels of Cav1.2 (Fig. 2A-G). However, knockdown of Cacna2d1 significantly decreased plasma membrane expression of Cav1.2 in both INS-1 832/13 cells (Fig. 2H, I and Fig. 3A, B) and mouse islet β cells (Fig. 3E and Suppl. Fig. 1A). This is in line with results from heterologous expression studies in Xenopus oocytes (Shistik, Ivanina et al. 1995). The antiepileptic and antinociceptive drug gabapentin (GBP) is structurally similar to the neurotransmitter GABA. Whereas GBP does not bind to GABA receptors, it has affinity for the α2δ-1 and α2δ-2 subunits and has been suggested to influence their trafficking, as well as that of the associated voltage-gated Ca2+ channels (VGCCs), e.g. Cav1.2. In neurons, long-term GBP treatment reduces the expression of VGCC in the plasma membrane, while increasing VGCC density in the recycling endosome compartment (Bauer, Tran-Van-Minh et al. 2010), leading to markedly reduced Ca2+ currents (Hendrich, Van Minh et al. 2008). In line with this view, we observe that Cacna2d1 silencing results in Cav1.2 channels being retained in the recycling endosome compartment (Fig. 2J, K, Fig. 3C, D and Fig. 3F, Suppl. Fig. 1B,). Hence, our results suggest that α2δ-1 is involved in trafficking of Cav1.2 in insulin-secreting β cells. As indicated above, α2δ subunits could affect Ca2+ signaling in excitable cells. Indeed, several studies showed that the α2δ-1 subunit increases the inactivation rate for both Cav1.2 (Felix, Gurnett et al. 1997, Sipos, Pika-Hartlaub et al. 2000) and Cav2.1 (Felix, Gurnett et al. 1997), which is in line 16

with a report showing that both α2δ-1 and α2δ-2 augment Cav1.2 and Cav2.3 current inactivation (Hobom, Dai et al. 2000). It was recently reported that α2δ-1 deletion in mouse resulted in decreased Ca2+ currents through all high-VGCC channels (Cav1 and Cav2 families) in pancreatic β-cells and thereby reduced insulin secretion and impaired glucose tolerance (Mastrolia, Flucher et al. 2017). However, the detailed cellular effects of α2δ-1 on trafficking of VGCC isoforms was not investigated. In the present study, we have extended on these data by exploring the effects of inhibiting α2δ-1 by RNA interference or GBP on depolarization-evoked increases in cytosolic free Ca2+ concentrations, voltage-gated Ca2+ currents, single-cell exocytosis and insulin secretion. Interestingly, single cell cytosolic Ca2+ signaling and voltage-gated Ca2+ currents were both strikingly decreased in INS-1 832/13 cells and mouse islet β cells by either Cacna2d1 silencing or 24 h exposure to GBP (Fig. 4A, B and C), furthermore, silencing of Cacna2d1 or/and Tcf7l2 pronouncedly decresed cytosolic Ca2+ under glucose stimulated condition (Fig. 5A-D). However, report showed that glucose-stimulated Ca2+ influx was not impaired in whole islet from Tcf7l2 null mice versus control until the animals were put on a high fat diet (Mitchell, Mondragon et al. 2015). The islet consists of α, β, δ, and pp cells which is complicated because of the communication between each other, and more importantly, they also observed a decreased β cell mass without changing of α cell number due to the deletion of Tcf7l2, which was contradictory for their observations. Nontheless, it is worthy of studying in islet β cells. Cacna2d1 silencing as expected decreased insulin secretion (Fig. 5E), whereas insulin secretion was completely unaffected in GBP-treated cells (not shown). The reason for this is not clear, but we could exclude any confounding effects by GBP acting directly on the exocytotic system (Suppl. Fig. 2). A possible explanation is that an alternative biological pathways affected by GBP that compensate the effect of α2δ subunit inhibition in insulin secretion. Such pathways include, but are not limited to, increase GABA synthesis (Taylor 1997, Kukkar, Bali et al. 2013). If acting on GABAA receptors, increased production of GABA will enhance beta cell membrane depolarization, prolong time for Ca2+ influx and increase insulin release (Maneuf, Gonzalez et al. 2003, Braun, Ramracheya et al. 2012).

17

This potential effect is overruled in voltage-clamped cells, but not in the insulin secretion experiments, which could explain the discrepant results. Support for GBP having additional insulinotropic effects comes from case report of severe hypoglycaemia in six (diabetic and non-diabetic) patients treated with GBP (Scholl, van Eekeren et al. 2015). Genome-wide association studies (GWAS) have consistently shown that common non-coding variation in the transcription factor 7-like 2 (TCF7L2) gene confer the strongest genetic risk for type 2 diabetes (T2D) (Cauchi, Meyre et al. 2006, Florez, Jablonski et al. 2006, Grant, Thorleifsson et al. 2006, Groves, Zeggini et al. 2006, Zhang, Qi et al. 2006, Florez 2007, Freathy, Weedon et al. 2007). TCF7L2 is important for maintaining the secretory function of mature β cells. Previous reports have shown that silencing of TCF7L2 results in decreased β-cell survival and attenuation of glucoseinduced insulin secretion (Shu, Sauter et al. 2008, Zhou, Zhang et al. 2012). It is very interesting to know how TCF7L2 affects insulin secretion. TCF7L2 is a transcriptional regulator controlled by the Wnt-signaling pathway (Smith 2007, Osmark, Hansson et al. 2009) and targets a number of genes, such as ISL1, MAFA, and PDX, which form a regulatory network and affect insulin secretion in rodent and human pancreatic islets (Zhou, Park et al. 2014). This can explain why silencing of Tcf7l2 further reduced insulin secretion compared to only silencing Cacna2d1. The consensus model of KATP channel closure triggering Ca2+ channels opening and Ca2+ influx, leading to insulin granule fusion with the plasma membrane in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process, including synaptobrevin/VAMP, SNAP-25, syntaxin, α-SNAP, and the putative Ca2+-sensing proteins synaptotagmin I and II (Rorsman, Eliasson et al. 2000, Gerber and Sudhof 2002). The synaptotagmin (Syt) gene family, comprising 15 members, has long been suggested as the Ca2+ sensor, mediating glucose-induced insulin secretion (Gauthier and Wollheim 2008, Andersson, Olsson et al. 2012). Both Tcfl7l2 and α2δ-1 regulate expression of Syt14, Stxbp1 and Vamp2 (Fig. 6D-F), however, downregulation of these genes by silencing of Tcf7l2 could not be rescued by overexpression of α2δ-1 (Fig. 6G-I). These results provide an explanation to why 18

overexpression of α2δ-1 fails to fully rescue the impaired insulin secretion in Tcf7l2-silenced cells. The latter failure occurred even though the glucose-induced cytosolic Ca2+ signaling was normalized by overexpression of α2δ-1 (Fig. 6A -C). In conclusion, in the present study we have identified Cacna2d1 as a Tcf7l2 target that affects insulin secretion by suppressing VGCC transport to the plasma membrane, thereby reducing Ca2+ signaling. These results underscore the value of detailed phenotyping e.g. when trying to decipher the effects of a genetic variant. Acknowledgements We thank Britt-Marie Nilsson and Anna-Maria Veljanovska Ramsay for expert technical assistance. Y.Y.’s position was financed by China Scholarship Council (201306310018). Grants to E.R. supporting this project include: the Swedish Research Council (2017-01090), Swedish Diabetes Association, Diabetes Wellness Foundation Sweden, and grants for clinical research (ALF). Grants to E.Z.: Swedish research council (2018-03258). The study used equipment/infrastructure funded by the Knut and Alice Wallenberg Foundation, the Swedish Foundation for Strategic Research (LUDC-IRC) and the strategic research area EXODIAB. Declarations of interest None Contribution statement E.R. and T.T. conceived the study. Y.Y, M.B, C.L, Y.Z, E.R. designed experiments, acquired and analysed data. E.R, T.T, E.Z, L.E and O.H analysed and interpreted data. Y.Y and A.K drafted the article. All authors revised the article and approved it for publishing. E.R is the guarantor of this work.

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Figure legends Fig 1. Silencing of Tcf7l2 reduces α2δ-1 expression A. mRNA expression of Tcf7l2 after silencing of Tcf7l2 (si-Tcf7l2) versus negative control (NC) in INS-1 832/13 cells; n = 3, paired Student’s t-tests ***p < 0.001. B. mRNA expression of Cacna2d1 after silencing of Tcf7l2 (si-Tcf7l2) versus negative control (NC) in INS-1 832/13 cells; n = 3, paired Student’s t-tests **p < 0.01. C. As (A), but in cultured rat islets; n = 3, paired Student’s t-tests **p < 0.01. D. As (B), but in cultured rat islets; n = 3, paired Student’s t-tests *p < 0.05. E. Change in α2δ-1 protein levels after silencing of Tcf7l2 versus negative control. F. The densitometry analysis for relative expression of α2δ-1 in each condition for E; n=4, unpaired Student’s t-tests **p < 0.01.

Fig 2. Silencing of Cacna2d1 does not affect expression of Cav1.2, but its membrane targeting

A. mRNA expression of Cacna2d1 after silencing of Cacna2d1 versus negative control in INS-1 832/13 cells; n = 3, paired Student’s t-tests ***p < 0.001. B. mRNA expression of Cacna1c after silencing of Cacna2d1 versus negative control in INS-1 832/13 cells; n = 3, ns: not signigicant. C. As (A), but in cultured rat islets; n = 3, paired Student’s t-tests ***p < 0.001. D. As (B), but in cultured rat islets; n = 3, ns: not signigicant. E. Reduced α2δ-1 protein and unchanged L-type Cav1.2 protein levels after silencing of Cacna2d1 versus negative control in whole INS-1 832/13 cell lysate. F-G.Densitometry analysis for E; n= 4, paired Student’s t-tests ****p < 0.0001, ns: not signigicant.

H. Reduced Cav1.2 protein levels after knockdown of Cacna2d1 in the PM fraction of INS-1 832/13 cells versus negative control. I. Presence of Cav1.2 at the PM after Cacna2d1 silencing in (H), analysed by densitometry and expressed in percent of the average value in negative control-treated cells, n = 3, paired Student’s t-tests *p < 0.05. J. Increased Cav1.2 protein levels after knockdown of Cacna2d1 in the endosome fraction of INS-1 832/13 cells versus negative control. K. Presence of Cav1.2 at the endosome after Cacna2d1 silencing in (J), analysed by densitometry and expressed in percent of the average value normalized to Rab11, Na+/K+-ATPase and SOD4, n = 3, paired Student’s t-tests *p < 0.05.

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Fig 3. Silencing of Cacna2d1 promotes the trafficking of Cav1.2 to the recycling endosome A. Immunostaining of Cav1.2 (green) and PM marker Na+/K+-ATPase (orange) in INS-1 832/13 cells after silencing Cacna2d1 (si-α2δ-1) for 48 h versus negative control (NC). B. Cav1.2 localization in (A) calculated as the mean intensity of Cav1.2 immunofluorescence in the PM over that in the cytosol; n = 24 cells for each group, unpaired Student’s t-tests **p < 0.01. C. Immunostaining of Cav1.2 and the recycling endosome marker Rab11 after knockdown of Cacna2d1 versus negative control. D. Average correlation R of Cav1.2 with Rab11 to assess colocalization in (C); n = 30, unpaired Student’s t-tests ***p < 0.001. E.

Cav1.2 localization with PM marker Na+/K+-ATPase calculated as in (B), but in mouse islet β cells after silencing Cacna2d1 for 72 h versus negative control. n =26 cells each group, unpaired Student’s t-tests **p < 0.01.

F. Cav1.2 colocalization with Rab11 calculated as in (D), but in mouse islet β cells after silencing Cacna2d1 for 72 h versus negative control, n = 19 cells in each groups, unpaired Student’s t-tests *p < 0.05. G. Immunostaining of Cav1.2 (green) and PM marker Na+/K+-ATPase (orange) in INS-1 832/13 cells after silencing Tcf7l2 (si-Tcf7l2) for 72 h (si-Tcf7l2 + Cherry) and overexpression of α2δ-1 (si-Tcf7l2 + OE α2δ-1 ) for 48h versus negative control (NC+Cherry). H. Cav1.2 localization in (G) calculated as the mean intensity of Cav1.2 immunofluorescence in the PM over that in the cytosol; n = 23, 30, 27 cells, respectively, Tukey's multiple comparisons test ***p < 0.001, ****p < 0.0001. Fig 4. Silencing of Cacna2d1 affects high K+-evoked Ca2+ signaling and voltage-gated Ca2+ currents A. Fluo-5F AM fluorescence signal ratio normalized to baseline [Ratio(F/F0)] before and following addition of a depolarizing buffer with 70 mM-K+ (arrow) in conditions of negative control-treated INS-1 832/13 cells (NC), after 48 h silencing of Cacna2d1 (si-α2δ-1), gabapentin-treated cells (24 h; GBP) and Cacna2d1-silenced cells treated with gabapentin (siα2δ-1+GBP). B. Average peak Ca2+ fluorescence under conditions in (A) as indicated; n = 35, 38, 35, 36 cells, respectively, Dunnett's multiple comparisons test, ****p < 0.0001,***p < 0.001, *p < 0.05. C. Current-voltage (I-V) relations normalized to cell size for whole-mouse β cell Ca2+ currents, comparing negative control treated cells to those silenced for Cacna2d1, treated with gabapentin, or both; n = 8, 9, 7, 6, respectively. Dunnett's multiple comparisons test *p < 0.05 (NC vs si-α2δ-1), ♯p< 0.05 (NC vs NC+GBP). 25

D. As in (A), but comparing conditions between silencing of Tcf7l2 (si-Tcf7l2) and negative control treated INS-1 832/13 cells. E.

Average peak Ca2+ fluorescence under conditions in (D) as indicated; n = 38, 37 cells in each group, unpaired Student’s t-tests **p < 0.01.

Fig 5. Silencing of Cacna2d1 affects glucose-stimulated Ca2+ signaling and exocytosis A. Fluo-5F AM fluorescence signal ratio normalized to baseline [Ratio(F/F0)] before and following addition of a high glucose buffer with 16.7 mMG (arrow) followed by high K+ stimulation in negative control-treated INS-1 832/13 cells (NC), after 72 h silencing of Cacna2d1 (si-α2δ-1). B. As (A), but after silencing of Tcf7l2 (si-Tcf7l2) versus negative control treated INS-1 832/13 cells. C. As (A), but after silencing of Cacna2d1 and Tcf7l2 (si-α+si-T) versus negative control treated INS-1 832/13 cells. D. Average of area under curve under conditions in (A, B, C) as indicated; n = 57, 51, 51, 56 cells respectively, Dunnett's multiple comparisons test, ****p < 0.0001. E.

Insulin secretion in INS-1 832/13 cells in low glucose (2.8 mMG) or high glucose (16.7 mMG) after silencing of Cacna2d1 or/and Tcf7l2 versus negative control, all the secretion values showed as normalized to NC in high glucose; n = 8, Tukey's multiple comparisons test ****p < 0.0001,***p < 0.001.

Fig 6. Overexpression of α2δ-1 partially compensates for silencing of Tcf7l2 A. Fluo-5F AM fluorescence signal ratio normalized to baseline [Ratio(F/F0)] before and following addition of a high glucose buffer with 16.7 mMG (arrow) followed by high K+ stimulation in mCherry-lentivirus-treated INS-1 832/13 cells (NC+Cherry), and α2δ-1 overexpressed INS-1 832/13 cells (OE α2δ-1). B. As (A), but comparing between conditions with or without overexpression of α2δ-1 in Tcf7l2silenced cells. C. Average of area under curve under conditions in (A, B) as indicated; n = 44, 47, 60, 52 cells, respectively, Tukey's multiple comparisons test, ****p < 0.0001, *p < 0.05. D. mRNA expression of Syt14 comparing in conditions of silencing Cacna2d1, silencing Tcf7l2 and silencing of both Cacna2d1 and Tcf7l2 vesus negative control, n= 6, Tukey's multiple comparisons test, ****p < 0.0001.

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E.

mRNA expression of Stxbp1 comparing in conditions of silencing Cacna2d1, silencing Tcf7l2 and silencing of both Cacna2d1 and Tcf7l2 vesus negative control, n= 6, Tukey's multiple comparisons test, ****p < 0.0001, ***p < 0.001, *p < 0.05.

F.

mRNA expression of Vamp2 comparing in conditions of silencing Cacna2d1, silencing Tcf7l2 and silencing of both Cacna2d1 and Tcf7l2 vesus negative control, n= 6, Tukey's multiple comparisons test, **p < 0.01.

G. mRNA expression of Syt14 comparing in conditions of silencing Tcf7l2, overexpression of α2δ-1 and overexpression of α2δ-1 after silencing Tcf7l2 vesus control lentivirus treatment, n= 4, Tukey's multiple comparisons test, ***p < 0.001, *p < 0.05. H. mRNA expression of Stxbp1 comparing in conditions of silencing Tcf7l2, overexpression of α2δ-1 and overexpression of α2δ-1 after silencing Tcf7l2 vesus control lentivirus treatment, n=4, Tukey's multiple comparisons test, **p < 0.01, *p < 0.05. I.

mRNA expression of Vamp2 comparing in conditions of silencing Tcf7l2, overexpression of α2δ-1 and overexpression of α2δ-1 after silencing Tcf7l2 vesus control lentivirus treatment, n=4, Tukey's multiple comparisons test, *p < 0.05.

J.

High K+-stimulated insulin secretion in INS-1 832/13 cells after silencing Tcf7l2, overexpression of α2δ-1 and overexpression of α2δ-1 with silencing Tcf7l2 vesus that in control lentivirus treated cells; n=5, Tukey's multiple comparisons test ****p < 0.0001, **p < 0.01.

K. Insulin secretion in INS-1 832/13 cells in low glucose (2.8mMG) or high glucose (16.7 mMG) after silencing Tcf7l2, overexpression of α2δ-1 and overexpression of α2δ-1 with silencing Tcf7l2 vesus that in control lentivirus treated cells; n=4, Tukey's multiple comparisons test, ****p < 0.0001, ***p < 0.001. L.

Insulin secretion in INS-1 832/13 cells in low glucose (2.8mMG) or high glucose (16.7 mMG) after silencing Tcf7l2 without lentivirous-Cherry; n=3, Sidak's multiple comparisons test, ****p < 0.0001.

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Highlights •

Tcf7l2 controls expression of Cacna2d1/α2δ-1

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Silencing of Cacna2d1 prevents trafficking of Cav1.2 to the plasma membrane

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Silencing of Cacna2d1 retains Cav1.2 in recycling endosomes

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Silencing of Cacna2d1 affects Ca2+ signaling and exocytosis

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Overexpression of α2δ-1 partially counteracts the effect of Tcf7l2 silencing