TWIK-1 is a member of the two-pore domain K þ (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. The physiological role of TWIK-1 has remained enigmatic because functional expression of TWIK-1 channels is elusive. Here we report that native TWIK-1 forms a functional channel at the plasma membrane of astrocytes. A search for TWIK-1-binding proteins led to the identification of TREK-1, another member of the K2P family. The TWIK-1/TREK-1 heterodimeric channel is formed via a disulphide bridge between residue C69 in TWIK-1 and C93 in TREK-1. Gene silencing demonstrates that surface expression of TWIK-1 and TREK-1 are interdependent. TWIK-1/TREK-1 heterodimers mediate astrocytic passive conductance and cannabinoidinduced glutamate release from astrocytes. Our study sheds new light on the diversity of K2P channels.

Anoctamin-1 (ANO1) acts as a Ca2+-activated Cl− channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis.

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Two-pore domain K+ (K2P) channels have been shown to modulate neuronal excitability. The physiological role of TWIK-1, the first identified K2P channel, in neuronal cells is largely unknown, and we reported previously that TWIK-1 contributes to the intrinsic excitability of dentate gyrus granule cells (DGGCs) in mice. In the present study, we investigated the coexpression of TWIK-1 and TASK-3, another K2P member, in DGGCs. Immunohistochemical staining data showed that TASK-3 proteins were highly localized in the proximal dendrites and soma of DGGCs, and this localization is similar to the expression pattern of TWIK-1. TWIK-1 was shown to associate with TASK-3 in DGGCs of mouse hippocampus and when both genes were overexpressed in COS-7 cells. shRNA-mediated gene silencing demonstrated that TWIK-1/TASK-3 heterodimeric channels displayed outwardly rectifying currents and contributed to the intrinsic excitability of DGGCs. Neurotensin–neurotensin receptor 1 (NT–NTSR1) signaling triggered the depolarization of DGGCs by inhibiting TWIK-1/TASK-3 heterodimeric channels, causing facilitated excitation of DGGCs. Taken together, our study clearly showed that TWIK-1/TASK-3 heterodimeric channels contribute to the intrinsic excitability of DGGCs and that their activities are regulated by NT–NTSR1 signaling.

Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat–containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression profiling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.