Histidine triad nucleotide-binding protein 1 (HINT1) regulates Ca2+ signaling in mouse fibroblasts and neuronal cells via store-operated Ca2+ entry pathway

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 Ca2+ signaling remain unclear. Here, we studied Ca2+ signaling in cultured embryonic fibroblasts derived from wild-type control and HINT1 knockout (KO) mice. The resting cytosolic Ca2+ level (measured with fura-2) was not altered in fibroblasts lacking HINT1. The stored Ca2+ evaluated by measuring peak amplitude of ATP (10 μM)-induced Ca2+ transients in Ca2+-free medium was significantly larger in HINT1 KO fibroblasts than in wild-type cells. Ca2+ influx after external Ca2+ 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 Ca2+ 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 Ca2+ influx, while ATP-evoked Ca2+ release was not changed. Thus the study demonstrates a novel function of HINT1 that involves the regulation of SOC-mediated Ca2+ entry pathway (Orai1 and STIM1), essential for regulation of cellular Ca2+ homeostasis.