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Reversion mutation of cDNA CA8-204 minigene construct produces a truncated functional peptide that regulates calcium release in vitro and produces profound analgesia in vivo

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Abstract

Intracellular calcium is critical in orchestrating neuronal excitability and analgesia. Carbonic anhydrase-8 (CA8) regulates intracellular calcium signaling through allosteric inhibition of neuronal inositol trisphosphate receptor 1 (ITPR1) to produce profound analgesia. Recently, we reported the “G” allele at rs6471859 represents cis-eQTL regulating alternative splicing of a 1697 bp transcript (CA8-204G) with a retained intron, alternative polyadenylation site and a new stop codon producing a functional 26 kDa peptide with an extended exon 3. In this study we show the reversion mutation (G to C) at rs6471859 within the CA8-204G expression vector also produced a stable 1697 bp transcript (CA8-204C) coding for a smaller peptide (~ 22 kDa) containing only the first three CA8 exons. Surprisingly, this peptide inhibited ITPR1 (pITPR1) activation, ITPR1-mediated calcium release in vitro; and produced profound analgesia in vivo. This is the first report showing CA8-204C codes for a functional peptide sufficient to regulate calcium signaling and produce profound analgesia.

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Acknowledgements

This work was funded in part from NINDS R21 NS105880 (RCL); NINDS 3R21NS105880-01S1 (RCL); and NIDCR DE022903 (RCL, ERM) and DoD W81XWH-19-1-0525 (RCL). We would like to thank the Department of Anesthesiology, Perioperative Medicine, and Pain Management, University of Miami Miller School of Medicine, Miami, Florida for additional support.

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Correspondence to Roy C. Levitt.

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335_2020_9848_MOESM1_ESM.tif

Electronic supplementary material 1 Fig S1. Enhanced expression of CA8-204C protein in NBL cells. (A) Immunostaining (IHC) using anti-V5 (V5- CA8-201MT, green) antibodies. (B) IHC using antibodies anti-V5 (V5-CA8-201, green). (C) IHC using anti-FLAG (FLAG-CA8-204C, green). IHC using NBL cells transfected with AAV8-viral vectors containing CA8-201MT, CA8-201 and CA8-204C shows overexpression of CA8-201 and CA8-204C. Expression was not detected after transfection with CA8-201MT. The histogram represents the comparison of overexpression of CA8-204 with CA8-201, wherein the positive cells versus total cells were quantified using one-way ANOVA with Bonferroni's post-hoc test (***P<0.001, **P<0.01, N=8 each gene group). (Scale: 35μm) (TIF 3183 kb)

335_2020_9848_MOESM2_ESM.tiff

Electronic supplementary material 2 Fig S2. Expression of ITPR1 in forskolin untreated NBL cells transfected with CA8-204C and CA8-201. Untreated NBL cells transfected with CA8-201-V5 and CA8-204C-FLAG vectors were harvested and western blotting was performed using anti-ITPR1 antibodies, normalized by anti-vinculin antibody (TIFF 1504 kb)

335_2020_9848_MOESM3_ESM.tif

Electronic supplementary material 3 Fig. S3. Forskolin-Untreated NBL cells transfected with CA8-201-V5 and CA8-204C-FLAG vectors were harvested and IHC was performed using anti-FLAG and anti-V5 antibodies (primary) and treated with Alexa Fluor (488) antibody (Scale: 35 μM) (TIF 1975 kb)

335_2020_9848_MOESM4_ESM.tiff

Electronic supplementary material 4 Fig. S4. Single channel Intracellular calcium (ATP dependent release) as represented in this figure was monitored by Fura-2 in NBL cells transfected with empty vector (negative control), CA8-201 and CA8-204C. Single channel recording were taken at 340nm (bound form of Ca2+). Data are taken from experiments illustrated in Figure. 4.(Scale: 40μM) (TIFF 1740 kb)

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Upadhyay, U., Zhuang, G.Z., Diatchenko, L. et al. Reversion mutation of cDNA CA8-204 minigene construct produces a truncated functional peptide that regulates calcium release in vitro and produces profound analgesia in vivo. Mamm Genome 31, 287–294 (2020). https://doi.org/10.1007/s00335-020-09848-y

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  • DOI: https://doi.org/10.1007/s00335-020-09848-y

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