Car8 dorsal root ganglion expression and genetic regulation of analgesic responses are associated with a cis-eQTL in mice
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Carbonic anhydrase-8 (Car8 mouse gene symbol) is devoid of enzymatic activity, but instead functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) to regulate this intracellular calcium release channel important in synaptic functions and neuronal excitability. Causative mutations in ITPR1 and carbonic anhydrase-8 in mice and humans are associated with certain subtypes of spinal cerebellar ataxia (SCA). SCA mice are genetically deficient in dorsal root ganglia (DRG) Car8 expression and display mechanical and thermal hypersensitivity and susceptibility to subacute and chronic inflammatory pain behaviors. In this report, we show that DRG Car8 expression is variable across 25 naïve-inbred strains of mice, and this cis-regulated eQTL (association between rs27660559, rs27706398, and rs27688767 and DRG Car8 expression; P < 1 × 10−11) is correlated with nociceptive responses in mice. Next, we hypothesized that increasing DRG Car8 gene expression would inhibit intracellular calcium release required for morphine antinociception and might correlate with antinociceptive sensitivity of morphine and perhaps other analgesic agents. We show that mean DRG Car8 gene expression is directly related to the dose of morphine or clonidine needed to provide a half-maximal analgesic response (r = 0.93, P < 0.00002; r = 0.83, P < 0.0008, respectively), suggesting that greater DRG Car8 expression increases analgesic requirements. Finally, we show that morphine induces intracellular free calcium release using Fura 2 calcium imaging in a dose-dependent manner; V5-Car8 WT overexpression in NBL cells inhibits morphine-induced calcium increase. These findings highlight the ‘morphine paradox’ whereby morphine provides antinociception by increasing intracellular free calcium, while Car8 and other antinociceptive agents work by decreasing intracellular free calcium. This is the first study demonstrating that biologic variability associated with this cis-eQTL may contribute to differing analgesic responses through altered regulation of ITPR1-dependent calcium release in mice.
We thank Drs. D. A. Lubarsky and K. Candiotti for their generous input and support. This work was funded by grants from the NIDCR R01 DE022903; the NIDA R031410; funding from Algynomics Inc. Chapel Hill, NC; and funding from the Department of Anesthesiology, Perioperative Medicine, and Pain Management, University of Miami Miller School of Medicine, Miami, Florida.
TW designed and ran the DRG eQTL studies and data analyses. GYZ, YK, and MO ran the NBL studies on morphine response and Car8 antagonism. LD, WM, SBS, ESF, KDS, DME, GYZ, and UU assisted with manuscript preparation, provided critical scientific review, revisions, and editing. ERM contributed to data analyses, reviewed and revised the manuscript. RCL designed the overall study, supervised the studies, setup collaborations, analyzed data, and wrote the manuscript.
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