Cellular Model for the Analysis of IRBIT-Dependent Regulation of the Type 1 IP3 Receptor

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Abstract

In vertebrate genomes, three genes encode subunits of IP3 receptors, including IP3R1, IP3R2, and IP3R3. Despite high homology between different subunits, homotetrameric IP3 receptors formed by IP3R1, IP3R2, and IP₃R3 in the endoplasmic reticulum membrane are markedly distinct by their functional features and regulatory mechanisms. It was particularly reported that IP3R1 is specifically regulated by the IP3R binding protein released with IP₃ (IRBIT), which competes with IP3 for binding to IP3R1. In turn, affinity of IRBIT/IP₃R1 binding is regulated by phosphorylation of IRBIT. By using the CRISPR/Cas9 approach to edit the genome of HEK-293 cells, two monoclonal cell lines were generated as a platform for uncovering a role of IRBIT and associated regulatory circuits in control of the IP₃R1 activity. In one line, HEK-IP3R1, IP₃R2, and IP3R3 genes were disrupted, while IP₃R1 was remained functional. Based on this line, the HEK-IP3R1/DIRBIT line was generated, wherein IRBIT (AHCYL1) gene was inactivated. The comparative analysis of ACh-induced Ca2+ signaling in cells of both lines was performed by employing the Ca2+ dye Fluo-4 and Ca2+ imaging. It was particularly shown that ACh mobilized Ca2+ in cells of both lines, which responded to the agonist at widely varied doses in an “all-or-nothing” manner. Yet, HEK-IP₃R1/DIRBIT cells turned out to be less sensitive to ACh compared to HEK-IP₃R1 cells.

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About the authors

E. Е. Kopylova

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

I. S. Masulis

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Author for correspondence.
Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

O. A. Rogachevskaja

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

E. N. Kochkina

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

Y. A. Kovalitskaya

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

M. F. Bystrova

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

S. S. Kolesnikov

Institute of Cell Biophysics, Russian Academy of Sciences, FRC PSCBR RAS

Email: irina.masulis@gmail.com
Russian Federation, Pushchino, Moscow oblast, 142290

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Supplementary files

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2. Table 2. Nucleotide sequences of target gene regions of the HEK-IP3R1/ΔIRBIT cell line after editing

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3. Fig. 1. Localization of protospacer (marked in blue) and PAM motifs (marked in red) on the sense and antisense DNA strands of exon 2 of the human IRBIT gene (NC_000001.11) for Cas9-10A nickase action.

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4. Fig. 2. a - Sequence of the wild-type IRBIT gene editing locus. b - Directed mutation of the donor matrix (single nucleotide substitution of C/T and ∆C) resulting in the disappearance of the antisense PAM and the appearance of a 645 bp XhoI CTCGAG site in the ssDNA donor.

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5. Fig. 3. a - Schematic of synthesized double-stranded matrix with mutations. b - Electrophoresis of amplified megaprimers AB (lane 1) and CD (lane 2). c -Electrophoresis of synthesized double-stranded dsDNA (640 bp) with point mutation (AD) and single-stranded matrix of donor ssDNA of the same nucleotide composition (differs from double-stranded matrix by migration rate and intensity of ethidium bromide staining).

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6. Fig. 4. Restriction analysis of the IRBIT locus (645 bp) amplified from genomic DNA isolated from cells transfected with CRISPR/Cas9-10A-ssDNA-donor components 72 h after transfection.

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7. Fig. 5. a - Localization of protospacers (underlined) on the sense and antisense strands of DNA of exon 2 of the human IRBIT gene (NC_000001.11) and schematic of their location. b - Schematic of amplification rounds to verify the obtained mutations in the edited region. c - Electrophoresis of eight representative monoclones obtained after transfection.

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8. Fig. 6. Ca2+ responses of cells to ACh. a - Representative monitoring of intracellular Ca2+ in HEK-IP3R1 (upper panel) (n = 366) and HEK-IP3R1/ΔIRBIT (lower panel) (n = 255) cells loaded with Fluo-4. The moments and durations of ACh applications at the indicated doses are indicated by horizontal lines above the experimental curves. The change in intracellular Ca2+ was assessed by the relative change in Fluo-4 fluorescence F/F0, where F=F-F0, F is the current fluorescence intensity, F0 is the mean fluorescence intensity at the initial time point of recording. b -Number of cells in each cell line generating Ca2+ responses to ACh at different agonist doses.

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