ß-COP Suppresses the Surface Expression of the TREK2.

K2P channels, also known as two-pore domain K+ channels, play a crucial role in maintaining the cell membrane potential and contributing to potassium homeostasis due to their leaky nature. The TREK, or tandem of pore domains in a weak inward rectifying K+ channel (TWIK)-related K+ channel, subfamily within the K2P family consists of mechanical channels regulated by various stimuli and binding proteins. Although TREK1 and TREK2 within the TREK subfamily share many similarities, ß-COP, which was previously known to bind to TREK1, exhibits a distinct binding pattern to other members of the TREK subfamily, including TREK2 and the TRAAK (TWIK-related acid-arachidonic activated K+ channel). In contrast to TREK1, ß-COP binds to the C-terminus of TREK2 and reduces its cell surface expression but does not bind to TRAAK. Furthermore, ß-COP cannot bind to TREK2 mutants with deletions or point mutations in the C-terminus and does not affect the surface expression of these TREK2 mutants. These results emphasize the unique role of ß-COP in regulating the surface expression of the TREK family.

ß-COP Regulates TWIK1/TREK1 Heterodimeric Channel-Mediated Passive Conductance in Astrocytes.

Mature astrocytes are characterized by a K+ conductance (passive conductance) that changes with a constant slope with voltage, which is involved in K+ homeostasis in the brain. Recently, we reported that the tandem of pore domains in a weak inward rectifying K+ channel (TWIK1 or KCNK1) and TWIK-related K+ channel 1 (TREK1 or KCNK2) form heterodimeric channels that mediate passive conductance in astrocytes. However, little is known about the binding proteins that regulate the function of the TWIK1/TREK1 heterodimeric channels. Here, we found that ß-coat protein (COP) regulated the surface expression and activity of the TWIK1/TREK1 heterodimeric channels in astrocytes. ß-COP binds directly to TREK1 but not TWIK1 in a heterologous expression system. However, ß-COP also interacts with the TWIK1/TREK1 heterodimeric channel in a TREK1 dependent manner and enhances the surface expression of the heterodimeric channel in astrocytes. Consequently, it regulates TWIK1/TREK1 heterodimeric channel-mediated passive conductance in astrocytes in the mouse brain. Taken together, these results suggest that ß-COP is a potential regulator of astrocytic passive conductance in the brain.

Anti-glioma effects of 2-aminothiophene-3-carboxamide derivatives, ANO1 channel blockers.

Anoctamin1 (ANO1), a calcium-activated chloride ion channel (CaCC), is associated with various physiological functions including cancer progression and metastasis/invasion. ANO1 has been considered as a promising target for cancer therapeutics as ANO1 is over-expressed in a variety of cancers including glioblastoma (GBM) and inhibition of ANO1 has been reported to suppress cell proliferation, migration and invasion in GBM. GBM is one of the most common and aggressive cancers with poor prognosis with median survival for 15 months. Lack of effective treatment options against GBM emphasizes urgent necessity of effective GBM therapeutics. In an effort to discover potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and synthesized a series of new 2-aminothiophene-3-carboxamide derivatives and performed SAR studies using both fluorescent cellular membrane potential assay and whole-cell patch-clamp recording. We observed that among these substances, 9c and 10q strongly suppress ANO1 channel activities and possess remarkable selectivity over ANO2. Unique structural feature of 10q, a cyclopentane-fused thiophene-3-carboxamide derivative, is the presence of benzoylthiourea functionality which dramatically contributes to activity. Both 9c and 10q suppress more strongly proliferation of GBM cells than four reference compounds including 3, Ani-9 and are also capable of inhibiting much more strongly colony formation than reference compounds in both 2D colony formation assay and 3D soft agar assay using U251 glioma cells. In addition, 9c and 10q suppress far more strongly migration/invasion of GBM cells than reference compounds. We, for the first time, found that the combination of ANO1 inhibitor (9c or 3) and temozolomide (TMZ) brings about remarkable synergistic effects in suppressing proliferation of GBM cells. Our study may provide an insight into designing selective and potent ANO1 inhibitors aiming at GBM treatment.

TTYH1 and TTYH2 Serve as LRRC8A-Independent Volume-Regulated Anion Channels in Cancer Cells.

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.