One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation.

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

A lack of effective treatment is one of the main factors contributing to gastric cancerrelated death. Discovering effective targets and understanding their underlying anticancer mechanism is key to achieving the best response to treatment and to limiting side effects. Although recent studies have shown that the cation channel transient receptor potential melastatin-2 (TRPM2) is crucial for cancer cell survival, the exact mechanism remains unclear, limiting its therapeutic potential. Here, using molecular and functional assays, we investigated the role of TRPM2 in survival of gastric cancer cells. Our results indicated that TRPM2 knockdown in AGS and MKN-45 cells decreases cell proliferation and enhances apoptosis. We also observed that the TRPM2 knockdown impairs mitochondrial metabolism, indicated by a decrease in basal and maximal mitochondrial oxygen consumption rates (OCRs) and ATP production. These mitochondrial defects coincided with a decrease in autophagy and mitophagy, indicated by reduced levels of autophagyand mitophagy-associated proteins (i.e. ATGs, LC3A/B II, and BNIP3). Moreover, we found that TRPM2 modulates autophagy through a JNK-dependent and mechanistic target of rapamycin (mTOR)-independent pathway. We conclude that in the absence of TRPM2, down-regulation of the JNK signaling pathway impairs autophagy, ultimately causing the accumulation of damaged mitochondria and death of gastric cancer cells. Of note, by inhibiting cell proliferation and promoting apoptosis, the TRPM2 down-regulation enhanced the efficacy of paclitaxel and doxorubicin in gastric cancer cells. Collectively, we provide compelling evidence that TRPM2 inhibition may benefit therapeutic approaches for managing gastric cancer.Gastric cancer is the fifth most common type of cancer worldwide, affecting millions each year (1-4). The five-year survival rate is estimated at approximately 30% (5) making it one of the deadliest malignancies in the world and the second leading cause of cancer-related mortality in Eastern Asia (6, 7). Currently, surgery is the most effective available therapy against gastric cancer; however, its efficacy is limited to the early-stage gastric cancer patients (8, 9). For patients with late-stage tumors, surgery is not an option and despite systemic chemotherapy, the disease is deemed incurable (10)(11)(12). Considering the poor efficacy of current anticancer agents, the increasing resistance to chemotherapy drugs and the lack of treatment options for late stage patients, the development of novel and effective therapeutic approaches is of critical importance.Over the last decade, Transient Receptor Potential (TRP) channels have gained considerable attention in the field of cancer-targeted therapy (13-16). TRP channels are often altered in cancer cells and disruption in their normal function can affect various signalling pathways, ultimately leading to cancer progression and growth (17, 18). The TRP family is divided into seven subfamilies consisting of a total of 28 members. Some of members inclu...

The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.

The calcium-sensing receptor (CaR), is a G protein-dependent receptor that responds to increments in extracellular Ca(2+) ([Ca(2+)](o)). We previously reported that an increase in [Ca(2+)](o) induced a release of intracellular calcium and Ca(2+) entry via store operated channels (SOCs). We also demonstrated that MCF-7 cells express Transient Receptor Potential canonical 1 (TRPC1) channels. Herein, we investigated CaR intracellular signaling pathways and examined the role of TRPC1 in CaR-induced cell proliferation, through the extracellular signal-regulated Kinases 1 & 2 (ERK1/2) pathways. Treatment by [Ca(2+)](o) increased both MCF-7 cell proliferation and TRPC1 expression. Both the [Ca(2+)](o) proliferative effect and TRPC1 protein levels were abolished by the ERK1/2 inhibitors. Moreover, [Ca(2+)](o) failed to increase cell proliferation either in the presence of CaR or TRPC1 siRNAs. Both [Ca(2+)](o) and the selective CaR activator spermine, elicited time and dose-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation was almost completely inhibited by treatment with the phospholipase C and the protein kinase C inhibitors. Treatment with 2-aminoethoxydiphenyl borate (2-APB), and SKF-96365 or by siTRPC1 diminished both [Ca(2+)](o)- and spermine-stimulated ERK1/2 phosphorylation. Moreover, down-regulation of TRPC1 by siRNA reduced the Ca(2+) entry induced by CaR activation. We conclude that the CaR activates ERK1/2 via a PLC/PKC-dependent pathway. Moreover, TRPC1 is required for the ERK1/2 phosphorylation, Ca(2+) entry and the CaR-proliferative effect.

Background:The cytoplasmic entrance to the CFTR channel pore is not defined. Results: Functionally important positively charged amino acid side chains attract Cl Ϫ to the cytoplasmic entrance to the pore.

The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.

Background: TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.