Structures of KcsA in complex with symmetrical quaternary ammonium compounds reveal a hydrophobic binding site.

Potassium channels allow for the passive movement of potassium ions across the cell membrane and are instrumental in controlling the membrane potential in all cell types. Quaternary ammonium (QA) compounds block potassium channels and have long been used to study the functional and structural properties of these channels. Here we describe the interaction between three symmetrical hydrophobic QAs and the prokaryotic potassium channel KcsA. The structures demonstrate the presence of a hydrophobic pocket between the inner helices of KcsA and provide insight into the binding site and blocking mechanism of hydrophobic QAs. The structures also reveal a structurally hidden pathway between the central cavity and the outside membrane environment reminiscent of the lateral fenestration observed in sodium channels that can be accessed through small conformational changes in the pore wall. We propose that the hydrophobic binding pocket stabilizes the alkyl chains of long-chain QA molecules and may play a key role in hydrophobic drug binding in general.

Hepatitis C virus activates interleukin-1ß via caspase-1-inflammasome complex.

Interleukin-1ß (IL-1ß) is a potent pro-inflammatory cytokine involved in the pathogenesis of HCV, but the sensors and underlying mechanisms that facilitate HCV-induced IL-1ß proteolytic activation and secretion remains unclear. In this study, we have identified a signalling pathway leading to IL-1ß activation and secretion in response to HCV infection. Previous studies have shown the induction and secretion of IL-1ß through the inflammasome complex in macrophages/monocytes. Here, we report for the first time the induction and assembly of the NALP3-inflammasome complex in human hepatoma cells infected with HCV (JFH-1). We demonstrate that activation of IL-1ß in HCV-infected cells involves the proteolytic processing of pro-caspase-1 into mature caspase-1 in a multiprotein inflammasome complex. Next, we demonstrate that HCV is sensed by NALP3 protein, which recruits the adaptor protein ASC for the assembly of the inflammasome complex. Using a small interfering RNA approach, we further show that components of the inflammasome complex are involved in the activation of IL-1ß in HCV-infected cells. Our study also demonstrates the role of reactive oxygen species in HCV-induced IL-1ß secretion. Collectively, these observations provide an insight into the mechanism of IL-1ß processing and secretion, which is likely to provide novel strategies for targeting the viral or cellular determinants to arrest the progression of liver disease associated with chronic HCV infection.

Activation of transcription factor Nrf2 by hepatitis C virus induces the cell-survival pathway.

Oxidative stress has been implicated in various human diseases, including the pathogenesis of hepatitis C virus (HCV). Previous studies have shown the induction of oxidative stress in cultured cells expressing HCV genes. The transcription factor Nrf2 is known to be activated in response to oxidative stress, but the mechanism of its activation is not clearly understood. In this study, we first determined the induction of Nrf2 and then investigated the mechanism of Nrf2 activation in human hepatoma cells infected with HCV (JFH-1). Our results showed the induction and nuclear translocation of Nrf2 in a time-dependent manner. The HCV-mediated activation of Nrf2 was abrogated in the presence of an antioxidant, PDTC (pyrrolidine dithiocarbamate), and a Ca(2+) chelator, BAPTA-AM [1,2-bis(aminophenoxy)ethane N,N,N,N-tetraacetic acid tetra(acetoxymethyl) ester], which suggests a role for both reactive oxygen species and Ca(2+) signalling in the Nrf2-activation process. By using inhibitors of cellular kinases, we showed further that HCV-mediated phosphorylation/activation of Nrf2 is mediated by the mitogen-activated protein (MAP) kinases p38 MAPK and janus kinase. We also observed enhanced phosphorylation of Akt and its downstream substrate Bad in HCV-infected cells. Furthermore, by using a small interfering RNA approach, our results suggest a potential role for HCV-mediated Nrf2 activation in the survival of HCV-infected cells, a condition favourable for liver oncogenesis. Taken together, these results provide an insight into the mechanisms by which HCV induces intracellular events relevant to chronic HCV infection.

Rac GTPase is a hub for protein kinase A and Epac signaling in endothelial barrier protection by cAMP.

Elevation in intracellular cAMP level has been associated with increased endothelial barrier integrity and linked to the activation of protein kinase A (PKA). Recent studies have shown a novel mechanism of cAMP-mediated endothelial barrier regulation via cAMP-dependent nucleotide exchange factor Epac1 and Rap1 GTPase. This study examined a contribution of PKA-dependent and PKA-independent pathways in the human pulmonary endothelial (EC) barrier protection by cAMP. Synthetic cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP), induced dose-dependent increase in EC transendothelial electrical resistance which was associated with activation of PKA, Epac/Rap1, and Tiam/Vav/Rac cascades and significantly attenuated thrombin-induced EC barrier disruption. Both specific Epac/Rap1 activator 8CPT-2Me-cAMP (8CPT) and specific PKA activator N(6)-benzoyl-adenosine-3',5'-cyclic monophosphate (6Bnz) enhanced EC barrier, suppressed thrombin-induced EC permeability, and independently activated small GTPase Rac. SiRNA-induced Rac knockdown suppressed barrier protective effects of both PKA and Epac signaling in pulmonary EC. Intravenous administration of either 6Bnz, or 8CPT, significantly reduced lung vascular leak in the murine model of lung injury induced by high tidal volume mechanical ventilation (HTV, 30 ml/kg, 4 h), whereas combined treatment with 6Bnz and 8CPT showed no further additive effects. This study dissected for the first time PKA and Epac pathways of lung EC barrier protection caused by cAMP elevation and identified Rac GTPase as a hub for PKA and Epac signaling leading to enhancement of lung vascular barrier.

Spin Labeling of Potassium Channels.

Potassium channels are the ion channels most extensively studied by structural techniques. Whereas high-resolution crystal structures have provided key insights into the molecular architecture of these channels, spin labeling studies have helped to unveil the dynamic structural aspects underlying their function. From a practical standpoint, the popularity of spin labeling studies of potassium channels lies in their small size and relative ease of overexpression. The inherent fourfold symmetry of most potassium channels has also greatly facilitated spin labeling studies. This chapter focuses on the overexpression, purification, spin labeling, and subsequent reconstitution of modified potassium channels. It will discuss the general methods used to produce a suitable spin-labeled potassium channel sample and highlight some of the common pitfalls that can occur along the way. At the end of the chapter, we provide detailed methods to produce spin-labeled samples of KcsA and KvAP, the two most commonly studied potassium channels.

Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium.

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.