Martin Gosling

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

Drug block of the hERG potassium channel: insight from modeling.

Many commonly used, structurally diverse, drugs block the human ether‐a‐go‐go‐related gene (hERG) K+ channel to cause acquired long QT syndrome, which can lead to sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in the development of safe drugs. To gain insight about the structure of hERG and the nature of drug block we have produced structural models of the channel pore domain, into each of which we have docked a set of 20 hERG blockers. In the absence of an experimentally determined three‐dimensional structure of hERG, each of the models was validated against site‐directed mutagenesis data. First, hERG models were produced of the open and closed channel states, based on homology with the prokaryotic K+ channel crystal structures. The modeled complexes were in partial agreement with the mutagenesis data. To improve agreement with mutagenesis data, a KcsA‐based model was refined by rotating the four copies of the S6 transmembrane helix half a residue position toward the C‐terminus, so as to place all residues known to be involved in drug binding in positions lining the central cavity. This model produces complexes that are consistent with mutagenesis data for smaller, but not larger, ligands. Larger ligands could be accommodated following refinement of this model by enlarging the cavity using the inherent flexibility about the glycine hinge (Gly648) in S6, to produce results consistent with the experimental data for the majority of ligands tested. Proteins 2007.

Activation of Human TRPC6 Channels by Receptor Stimulation

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K0.5 of ∼10 μm and a Hill coefficient of 3–4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30–300 μm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 μm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.

F-actin Capping (CapZ) and Other Contractile Saphenous Vein Smooth Muscle Proteins Are Altered by Hemodynamic Stress A PROTEOMIC APPROACH

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3–10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 μm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser82 and Ser15Ser82 isoforms (p = 0.018). The abundance of actin-capping protein α2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC50 ∼50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = α1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED50 = 3 μg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis

Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The 2‐D system comprised a nonlinear immobilised pH 3–10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3–10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14 000–200 000 Daltons). Using a combination of peptide mass fingerprinting by matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins.

Insight into the mechanism of inactivation and pH sensitivity in potassium channels from molecular dynamics simulations

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.

Expression of Transient Receptor Potential C6 Channels in Human Lung Macrophages

Chronic obstructive pulmonary disease (COPD) is associated with pulmonary inflammation with increased numbers of macrophages located in the parenchyma. These macrophages have the capacity to mediate the underlying pathophysiology of COPD; therefore, a better understanding of their function in chronic inflammation associated with this disease is vital. Ion channels regulate many cellular functions; however, their role in macrophages is unclear. This study examined the expression and function of transient receptor potential (TRP) channels in human macrophages. Human alveolar macrophages and lung tissue macrophages expressed increased mRNA and protein for TRPC6 when compared with monocytes and monocyte-derived macrophages. Moreover, TRPC6 mRNA expression was significantly elevated in alveolar macrophages from patients with COPD compared with control subjects. There were no differences in mRNA for TRPC3 or TRPC7. Although mRNA for TRPM2 and TRPV1 was detected in these cells, protein expression could not be determined. Fractionation of lung-derived macrophages demonstrated that TRPC6 protein was more highly expressed by smaller macrophages compared with larger macrophages. Using whole-cell patch clamp electrophysiology, TRPC6-like currents were measured in both macrophage subpopulations with appropriate biophysical and basic pharmacological profiles. These currents were active under basal conditions in the small macrophages. These data suggest that TRPC6-like channels are functional on human lung macrophages, and may be associated with COPD.

Arterial flow conditions downregulate thrombomodulin on saphenous vein endothelium

BACKGROUND The antithrombogenic properties of venous endothelium may be attenuated when vein is implanted in the arterial circulation. Such changes may facilitate thrombosis, which is the final common pathway for saphenous vein arterial bypass graft occlusion. METHODS AND RESULTS Using human saphenous vein in a validated ex vivo flow circuit, we investigated (1) the possibility that arterial flow conditions (mean pressure, 100 mm Hg, 90 cpm, approximately 200 mL/min) alter the concentration of proteins involved in regulating thrombosis at the vessel wall and (2) the influence of ion channel blockade on such effects. Concentrations of thrombomodulin and tissue factor were quantified by Western blotting (ratio of von Willebrand factor staining) and immunohistochemistry (as a percentage of CD31-staining area). Thrombomodulin concentrations after 90 minutes of venous and arterial flow conditions were quantified by immunostaining (68.9+/-4.8% and 41.0+/-3.0% CD31, respectively; P<0.01) and by Western blotting (1.35+/-0.20 and 0. 15+/-0.03 ratio of von Willebrand factor, respectively; P<0.01). The ability of endothelial cells to generate activated protein C also decreased from 62+/-14 to 19+/-10 ng. min-1. 1000 cells-1 (P=0.01). The significant reduction in thrombomodulin was attenuated if calcium was removed from the perfusate but not by external vein stenting. Inclusion in the vein perfusate of drugs that reduce calcium entry (including Gd3+, to block stretch-activated ion channels, and nifedipine) abolished the reduction in thrombomodulin concentration observed after arterial flow conditions. In freshly excised vein, negligible concentrations of tissue factor were detected on the endothelium and concentrations did not increase after 90 minutes of arterial flow conditions, although the inclusion of nifedipine caused the immunostaining to increase from 3.0+/-0.4% to 8.5+/-0.7% CD31 (P<0.02). CONCLUSIONS In saphenous vein endothelium exposed to arterial flow conditions, there is rapid downregulation of thrombomodulin, sufficient to limit protein C activation, by a calcium-dependent mechanism.

TRP channels in airway smooth muscle as therapeutic targets

Cation channels are of fundamental importance in regulating the function of airway smooth cells especially bronchoconstriction in response to spasmogens, and are therefore key players in the pathogenesis of asthma. To date, the identity of these cation channels remains a mystery. However, the recently emerged transient receptor potential (TRP) cation channel family has provided several promising channel candidates. The identification of the key TRP channels involved in regulating airway smooth muscle contractility, and therefore airway tone, could provide new and exciting prospects for the development of novel therapies for the treatment of airway diseases such as asthma.