Glioma-specific cation conductance regulates migration and cell cycle progression.

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.

Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes.

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.

Amiloride docking to acid-sensing ion channel-1.

Amiloride is a small molecule diuretic, which has been used to dissect sodium transport pathways in many different systems. This drug is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as sodium/hydrogen antiporters and sodium/calcium exchangers. The exact structural basis for these interactions has not been elucidated as crystal structures of these proteins have been challenging to obtain, though some involved residues and domains have been mapped. This work examines the interaction of amiloride with acid-sensing ion channel-1, a protein whose structure is available using computational and experimental techniques. Using molecular docking software, amiloride and related molecules were docked to model structures of homomeric human ASIC-1 to generate potential interaction sites and predict which analogs would be more or less potent than amiloride. The predictions made were experimentally tested using whole-cell patch clamp. Drugs previously classified as NCX or NHE inhibitors are shown to also inhibit hASIC-1. Potential docking sites were re-examined against experimental data to remove spurious interaction sites. The voltage sensitivity of inhibitors was also examined. Using the aggregated data from these computational and experimental experiments, putative interaction sites for amiloride and hASIC-1 have been defined. Future work will experimentally verify these interaction sites, but at present this should allow for virtual screening of drug libraries at these putative interaction sites.

Proteolytic cleavage of human acid-sensing ion channel 1 by the serine protease matriptase.

Acid-sensing ion channel 1 (ASIC1) is a H(+)-gated channel of the amiloride-sensitive epithelial Na(+) channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical alphabetagammaENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems.

A truncated CFTR protein rescues endogenous DeltaF508-CFTR and corrects chloride transport in mice.

Cystic fibrosis (CF) is most frequently associated with deletion of phenylalanine at position 508 (DeltaF508) in the CF transmembrane conductance regulator (CFTR) protein. The DeltaF508-CFTR mutant protein exhibits a folding defect that affects its processing and impairs chloride-channel function. This study aimed to determine whether CFTR fragments approximately half the size of wild-type CFTR and complementary to the portion of CFTR bearing the mutation can specifically rescue the processing of endogenous DeltaF508-CFTR in vivo. cDNA encoding CFTR fragments were delivered to human airway epithelial cells and mice harboring endogenous DeltaF508-CFTR. Delivery of small CFTR fragments, which do not act as chloride channels by themselves, rescue DeltaF508-CFTR. Therefore, we can speculate that the presence of the CFTR fragment, which does not harbor a mutation, might facilitate intermolecular interactions. The rescue of CFTR was evident by the restoration of chloride transport in human CFBE41o- bronchial epithelial cells expressing DeltaF508-CFTR in vitro. More important, nasal administration of an adenovirus expressing a complementary CFTR fragment restored some degree of CFTR activity in the nasal airways of DeltaF508 homozygous mice in vivo. These findings identify complementary protein fragments as a viable in vivo approach for correcting disease-causing misfolding of plasma membrane proteins.

Generalizing on best practices in image processing: a model for promoting research integrity: Commentary on: Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images.

Modifying images for scientific publication is now quick and easy due to changes in technology. This has created a need for new image processing guidelines and attitudes, such as those offered to the research community by Doug Cromey (Cromey 2010). We suggest that related changes in technology have simplified the task of detecting misconduct for journal editors as well as researchers, and that this simplification has caused a shift in the responsibility for reporting misconduct. We also argue that the concept of best practices in image processing can serve as a general model for education in best practices in research.

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function.

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

The ups and downs of peer review.

This article traces the history of peer review of scientific publications, plotting the development of the process from its inception to its present-day application. We discuss the merits of peer review and its weaknesses, both perceived and real, as well as the practicalities of several major proposed changes to the system. It is our hope that readers will gain a better appreciation of the complexities of the process and, when serving as reviewers themselves, will do so in a manner that will enhance the utility of the exercise. We also propose the development of an international on-line training program for accreditation of potential referees.

Epithelial sodium channel in planar lipid bilayers.

Amiloride-sensitive Na+ channels belong to the epithelial Na+ channel (ENaC)-degenerin superfamily of ion channels. In addition to their key role in sodium handling, they serve diverse functions in many tissues. Improper functioning of ENaC has been implicated in several diseases, including salt-sensitive hypertension (Liddle's syndrome), salt-wasting syndrome (pseudohypoaldosteronism type I), pulmonary edema, and cystic fibrosis. We have utilized planar lipid bilayers, a well-defined system that allows simultaneous control of "internal" and "external" solutions, to study ENaCs.

Membrane transplantation to correct integral membrane protein defects.

In this report we show that the tendency of certain viruses to carry host membrane proteins in their envelopes can be harnessed for transplantation of small patches of plasma membrane, including fully functional, polytopic ion channel proteins and their regulatory binding partners. As a stringent model we tested the topologically complex epithelial ion channel CFTR. Initially an attenuated vaccinia virus was found capable of transferring CFTR in a properly folded, functional and regulatable form to CFTR negative cells. Next we generated viruslike particles (VLPs) composed of retroviral structural proteins that assemble and bud at the host cell plasma membrane. These particles were also shown to mediate functional ion channel transfer. By testing the capacity of complex membrane proteins to incorporate into viral envelopes these experiments provide new insight into the permissiveness of viral envelopment, including the ability of incorporated proteins to retain function and repair defects at the cell surface, and serve as a platform for studies of ion channel and membrane protein biochemistry.

Ethics and scientific publication.

This article summarizes the major categories of ethical violations encountered during submission, review, and publication of scientific articles. We discuss data fabrication and falsification, plagiarism, redundant and duplicate publication, conflict of interest, authorship, animal and human welfare, and reviewer responsibility. In each section, pertinent historical background and citation of relevant regulations and statutes are provided. Furthermore, a specific case(s) derived from actual situations is(are) presented. These cases were chosen to highlight the complexities that investigators and journals must face when dealing with ethical issues. A series of discussion questions follow each case. It is our hope that by increasing education and awareness of ethical matters relevant to scientific investigation and publication, deviations from appropriate conduct will be reduced.

Spontaneous autoinflation of saline mammary implants: further studies.

BACKGROUND: Previous studies have reported a hyperinflation of saline-filled breast implants. On removal, the implant fluid had changed from clear saline to a yellowish-brown color, with a viscous consistency similar to serum. OBJECTIVE: Our objective was to identify further the components of saline from implants that had undergone spontaneous autoinflation. Our hypothesis was that if serum albumin is present in the fluid, then other proteins would likely be found. METHODS: To screen and identify proteins in implant fluid, we used a proteomics-based approach that included 1- and 2-dimensional gel electrophoresis and mass spectrometry of protein samples. RESULTS: Four known proteins and 1 unknown protein product were identified. Based on 2-dimensional gel electrophoresis and mass spectrometry, 2 general observations can be made about the saline from the autoinflated implants: serum albumin was the most prevalent protein, and there are a large number of proteins that remain to be identified. CONCLUSIONS: There are multiple macromolecules that cross into the lumen of the prosthesis. We believe spontaneous autoinflation is occurring more often than is believed or reported.

Molecular cloning and characterization of human acid sensing ion channel (ASIC)2 gene promoter.

Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na(+)-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (-1551 to +117) was cloned and characterized. The ASIC2 promoter lacked a canonical TATA box, but contained one putative CCAAT box. Nucleotide sequencing of the promoter revealed the presence of a number of transcription factor-binding sites and a 404 bp CpG island upstream the transcription start site. Nested deletion mutants and transfection results showed that the construct between -133 and +117 base pairs conferred basal transcription specific activity. Mutation of Sp1 and CP2 sites in this region resulted in a 70 and 95% decrease in promoter activity, respectively. Gel shift assays demonstrated the existence of specific protein binding to the SP1 and CP2 elements. There was no mutation in the CpG island in six glioma cell lines, but methylation-specific PCR showed methylation in some of glioma cell lines and tumor tissues, and treatment with the methylation inhibitor 5-Aza-2'-deoxycytidine could partially restore ASIC2 expression in cell lines, suggesting that epigenetic mechanisms may contribute to dysregulated ASIC2 expression.

Methods to study CFTR protein in vitro.

CFTR is a cyclic AMP and nucleotide-related chloride-selective channel with a low unitary conductance. Many of the physiological roles of CFTR are effectively studied in intact cells and tissues. However, there are also several clear advantages to the application of cell-free technologies to the study of the biochemical and biophysical properties of CFTR. When expressed in heterologous cells, CFTR is processed relatively poorly, depending, however, on the cell-type analysed. In some cells, only 20-25% of the protein which is initially synthesized exits the endoplasmic reticulum to insert into the cell membrane [Cell 83 (1995) 121; EMBO J. 13 (1994) 6076]. Further, many of the disease-causing mutants of CFTR result in even lower processing efficiencies. Therefore, several procedures have been developed to study regulated CFTR channel function expressed in microsomal membranes and following its purification and reconstitution. These experimental approaches and their application are discussed here.

The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl- channel.

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.