title: Histidine triad nucleotide-binding protein 1 (HINT1) regulates Ca(2+) signaling in mouse fibroblasts and neuronal cells via store-operated Ca(2+) entry pathway.
abstract: Recent findings indicate that histidine triad nucleotide-binding protein 1 (HINT1) is implicated in the pathophysiology of certain psychiatric disorders and also exhibits tumor suppressor properties. However, the authentic functions of HINT1 in cellular physiology and especially its role in Ca(2+) signaling remain unclear. Here, we studied Ca(2+) signaling in cultured embryonic fibroblasts derived from wild-type control and HINT1 knockout (KO) mice. The resting cytosolic Ca(2+) level (measured with fura-2) was not altered in fibroblasts lacking HINT1. The stored Ca(2+) evaluated by measuring peak amplitude of ATP (10 μM)-induced Ca(2+) transients in Ca(2+)-free medium was significantly larger in HINT1 KO fibroblasts than in wild-type cells. Ca(2+) influx after external Ca(2+) restoration, likely via store- and receptor-operated channels (SOCs and ROCs, respectively), was greatly (by 2-fold) reduced in HINT1 KO fibroblasts. This correlated with a downregulated expression of Orai1 and stromal interacting molecule 1 (STIM1), essential components of store-operated Ca(2+) entry pathway. Expression of canonical transient receptor potential (TRPC)3 and TRPC6, which function as ROCs, was not altered in HINT1 KO fibroblasts. Immunoblots also revealed that Orai1 was downregulated by twofold in brain lysates of HINT1 KO mice compared with the wild-type littermates. Importantly, silencer RNA knockdown of HINT1 in Neuro-2A cells markedly downregulated Orai1 and STIM1 protein expression and significantly (by 2.5-fold) reduced ATP-induced Ca(2+) influx, while ATP-evoked Ca(2+) release was not changed. Thus the study demonstrates a novel function of HINT1 that involves the regulation of SOC-mediated Ca(2+) entry pathway (Orai1 and STIM1), essential for regulation of cellular Ca(2+) homeostasis.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Cristina I
authors.initials: CI
authors.lastname: Linde
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.forename: Jia Bei
authors.initials: JB
authors.lastname: Wang
authors.forename: Vera A
authors.initials: VA
authors.lastname: Golovina
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D000071740:ORAI1 Protein; D034741:RNA, Small Interfering; D000071737:Stromal Interaction Molecule 1
chemical_list: D015220:Calcium Channels; C489275:Hint1 protein, mouse; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; D034741:RNA, Small Interfering; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1
keywords: HINT1 knockout mice; Orai1; STIM1; TRPC proteins; receptor-operated Ca2+ entry