Efhc1- and Ip₃r1-deficient mice used for this study have been developed and reported previously [3, 24]. The heterozygous mice were maintained on C57BL/6J background. Heterozygous knockout Efhc1^+/– or Ip₃r1^+/– mice were interbred to obtain wild-type (WT), heterozygous, and homozygous knockout mice. Food and water were available ad libitum, and cages (of less than 5 animals) were kept at 23°C on a 12-h/12-h light/dark cycle, with the lights off at 20:00. When euthanasia of mice was necessary, cervical dislocation was performed in accordance with institutional guidelines.

Mouse 6A3-mAb was reported previously [2, 4]. Following antibodies were also used: IP₃R1 [KM1112 [26], 18A10 [24], 10A6 [27], H-80 or C-20 (Santa Cruz Biotechnology, USA)], IP₃R2 (KM1083), IP₃R3 (KM1082 [26] and BD Transduction lab, USA), FLAG (Sigma, USA), Myc (Cell Signaling Technology, USA), GAPDH (Santa Cruz Biotechnology), PRKCSH (Santa Cruz Biotechnology), and DsRed (Invitrogen).

Mice were deeply anesthetized with three types of mixed anesthetic agents (0.3 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol) and perfused intracardially with 0.9% NaCl, followed by 4% paraformaldehyde (TAAB, UK) in phosphate buffered saline (PBS). Preparations of mouse sagittal brain sections (paraffin) from embryonic day 14 (E14), unknown sex, and postnatal day 15 (P15) male mice and immunohistochemical staining were carried out as described previously [2, 3]. The 18A10 antibody for IP₃R1 and 6A3-mAb for myoclonin1 were used for staining. Colorimetric and fluorescence images were acquired using the AX80 light-microscope (Olympus, Japan) and TCS SP2 confocal laser scanning microscope (Leica, Germany), respectively.

EFHC1 clone was described previously [1]. We amplified parts of IP₃R1 (GenBank: NM_002222), IP₃R2 (NM_002223), IP₃R3 (NM_002224), and PRKCSH (NM_002743) from human adult brain cDNA by PCR and cloned them into pcDNA3-MycN, pcDNA3-FlagN, or pcDNA3-monomeric red fluorescence protein (mRFP) vectors. We introduced mutations by using QuickChange Site-Directed Mutagenesis kit (Agilent Technologies, USA) and confirmed nucleotide changes as well as integrity of full sequences by DNA sequencing.

Mouse embryonic fibroblasts (MEFs) were prepared as described previously from E16 unknown sex mouse tail [28]. Once the cells were confluent in poly-L-Lysine coated 60 mm dish (IWAKI, Japan), it was stored at –80°C until needed. To obtain glial cells, culture medium of hippocampal neuron culture prepared from E16 unknown sex mouse brain [1, 2] was replaced with Dulbecco’s Modified Eagle Medium (D-MEM) + 10% fetal bovine serum (FBS) + 30 U/mL penicillin and 30 mg/mL streptomycin (P/S) at 2 days in vitro (DIV) and cultured for 18 DIV. During the culture of MEFs or glial cells, visual inspection revealed no differences in cell viability or morphology between cells derived from WT and Efhc1^–/– mice. The glial cells were harvested and stored in –80°C for subsequent assay. MEFs and HeLa.S3 cells were transfected with expression constructs using Lipofectamine LTX and PLUS reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. Briefly, the cells were exposed to the lipid-DNA complex in serum-free medium (Opti-MEM I; Thermo Fisher Scientific). Subsequently, the cells were incubated in D-MEM supplemented with 10% FBS. Transfected cells were analyzed 24–48 hours post-transfection. The HeLa.S3 cell line was originally obtained from the American Type Culture Collection and cultured under standard conditions. Cells were authenticated by short tandem repeat profiling and were free of mycoplasma contamination.

We washed transfected HeLa.S3 cells twice in PBS and lysed in lysis buffer [10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 or 1% NP40 and supplemented with protease inhibitor (Complete, Roche, Switzerland)]. Co-immunoprecipitation (co-IP) studies were carried out as described previously [1]. We probed blot with anti-FLAG, anti-Myc, or anti-DsRed antibodies and developed it with Western Lightning kit (Perkin Elmer, USA). Antibodies, IP₃R1 (KM1112, 10A6, H-80 or C-20, Santa Cruz Biotechnology), IP₃R2 (KM1083), IP₃R3 (KM1082 and BD Transduction lab), PRKCSH (Santa Cruz Biotechnology), myoclonin1 (6A3-mAb), and GAPDH (Santa Cruz Biotechnology) were used for western blot analysis. Wherever necessary, we stripped blots and re-probed with respective antibodies.

Ca²⁺ imaging analyses were done as described previously [29]. We placed the cells on an inverted microscope IX70 (Olympus) and observed through an objective lens UApoN40XO340 (Olympus), etc. Some of the images were acquired using Olympus IX81-ZDC. The cells were illuminated by a xenon lamp through excitation filters, 340AF15 (Omega, USA) and 380HT15 (Omega), alternately. We used a computer-controlled filter exchanger Lambda 10-2 (Sutter, USA) to switch filters. We used a dichroic mirror and an emission filter 430DCLP (Omega) and 510WB40 (Omega), respectively. To eliminate Ca²⁺ influx, we performed all experiments in absence of extracellular Ca²⁺. We measured IICR with addition of 5 µM histamine [30] for HeLa.S3 cells or 300 nM bradykinin (BK) [31] for MEFs, whereas [Ca²⁺]_ER was measured with addition of 5 µM ionomycin [32]. All measurements shown were representative results from two to four independent experiments (used 3 dishes per condition were used in each experiment).

Data were presented as mean ± standard error of the mean (s.e.m.) and statistical significance of differences between means was tested using unpaired t-test. Statistical significance levels were defined as P < 0.05.

Immunohistochemical analyses revealed that myoclonin1 and IP₃R1 were abundantly expressed in choroid plexus at E14 (Figure 1A–C). As reported previously [2], the expression of myoclonin1 in choroid plexus was very transient, appearing at embryonic stages (E14–18) and gradually switching off until P15 (Figure 1D). In contrast, the IP₃R1 expression in choroid plexus was observed at E14 (Figure 1B and C) and postnatal period (Figure 1D), suggesting its continuous expression through embryonic and postnatal stages. At P15, myoclonin1 was prominently expressed in motile cilia of ependymal cells but also moderately in cell body (Figure 1D); the somatic expression was more prominent in the 3rd ventricle as reported previously [2]. The IP₃R1 expression was mainly observed in the somata and not in the cilia of ependymal cells (Figure 1D). Altogether, these results indicate that myoclonin1 and IP₃R1 proteins are co-expressed in embryonic choroid plexus and postnatal ependymal cells.

We next investigated whether myoclonin1 binds to IP₃Rs. Because a number of proteins interact with either N- or C-terminal cytosolic regions of IP₃R1 and modulate its activity [19], we selected these regions to test their ability to interact with myoclonin1 for co-IP assay. It actually revealed that myoclonin1 binds to the C-terminal region of IP₃R1 but not to the N-terminal region or to Endophilin, a randomly selected protein with a molecular weight of 50 kDa used as a negative control (Figure 2A, B and Figure S1). Using a series of C-terminal deletion constructs, we further narrowed down the interacting region to amino acid residues (a.a.) 2565–2625 by co-IP assay (Figure 2A and C). Inversely, co-IP analyses using a series of deletion fragments of myoclonin1 revealed that each of the three DM10 domains of myoclonin1 independently bound to IP₃R1 (Figure 2D and E). C-terminal regions of three IP₃R subtypes (IP₃R1, IP₃R2, IP₃R3) are highly conserved (Figure S2A), and a co-IP assay revealed that all C-termini of these IP₃R subtypes similarly bound to myoclonin1 (Figure S2B).

PRKCSH is a gene that encodes the PRKCSH, also known as 80K-H, involved in protein folding in the ER. A previous report showed that PRKCSH was identified as an interacting protein of IP₃Rs by yeast two-hybrid screening and regulates its activity [25]. Because binding sites of IP₃R1 for PRKCSH and myoclonin1 look overlapping, we investigated interactions among these three proteins. As previously reported, co-IP revealed that a.a. 2555–2594 of IP₃R1 bound to PRKCSH (Figure S3). A co-IP of myoclonin1 and a series of PRKCSH deletion constructs, including two clones, a.a. 32–528 and 184–528, which were identified as fragments binding to IP₃R1 [25] and additional newly designed one, revealed that a.a. 400–448 of PRKCSH binds to myoclonin1 (Figure 3A–E and Figure S1). Similarly to IP₃R1 (Figure 2D and E), the three DM10 domains of myoclonin1 independently bound to PRKCSH (Figure 3F and G).

We subsequently measured levels of [Ca²⁺]_ER in MEFs and glial cells derived from Efhc1^–/– and WT littermates by applying ionomycin, an ionophore that induces formation of Ca²⁺-permeable pores in cellular membranes, leading to complete emptying of [Ca²⁺]_ER independently of IP₃Rs activation [32]. The assay showed that [Ca²⁺]_ER levels of the Efhc1^–/– cells were significantly higher compared to WT (Figure 4A and B). We also measured IICR by addition of BK, which stimulates phospholipase C (PLC) metabolism of phosphatidylinositol-4,5-bisphosphate (PIPP) to IP₃ [31], and observed a higher IICR level in Efhc1^–/– MEFs than in WT (Figure 4C). Western blot analyses revealed that myoclonin1 expression was abrogated in the Efhc1^–/– MEFs, while those of IP₃Rs and PRKCSH remained unchanged (Figure S4). These results indicate that myoclonin1 deficiency enhances [Ca²⁺]_ER and IICR.

We further investigated whether myoclonin1 reduces [Ca²⁺]_ER and IICR. Human HeLa.S3 cells were transfected with mRFP-fused myoclonin1, which did not affect expression of IP₃Rs and PRKCSH (Figure S5A). Ca²⁺ imaging revealed that [Ca²⁺]_ER by applying ionomycin and IICR by histamine, which generates IP₃ through activation of PLC [30], were significantly decreased by myoclonin1 over-expression (Figure S5B and S5C). To confirm the effect, myoclonin1 was further re-introduced into Efhc1^–/– MEFs. Ca²⁺ imaging also revealed that both [Ca²⁺]_ER by ionomycin and IICR by BK were significantly attenuated in mRFP-myoclonin1 expressing MEFs compared to control one (Figure S5D and S5E). Together, these results indicate that myoclonin1 lowers [Ca²⁺]_ER and is involved in maintenance of ER-Ca²⁺ homeostasis.