Effects of phosphorylation on ion channel function

There is considerable evidence suggesting that intracellular second messengers can modulate the activity of ion channels, and that protein phosphorylation by the different protein kinases is a frequent intermediary in these modulatory effects. This conclusion, namely, that ion channel proteins are indeed substrates for phosphorylation, has been verified in numerous biochemical studies [reviewed in 1–6].The functional correlates of channel phosphorylation are known to involve a change in channel open probability and, in the case of voltage-sensitive ion channels, a shift in the voltage dependence of channel activation. The voltage dependence of ion channel gating appears to be governed by movement of charge in the voltage-sensing moiety. Analogous to alterations in enzyme activities following biochemical modification, phosphorylation of ion channel proteins may lead to conformational changes that subsequently alter their gating and/or conductive properties, giving rise to the observed changes in electrical activity. However, in many cases, it is not yet clear whether it is the ion channels themselves that are directly modified, or whether phosphorylation is simply an early step in a cascade of events that leads ultimately to modulation of channel activity. The development and application of single-channel recording techniques in membrane patches and in artificial planar lipid bilayers has provided a means to investigate the effects of phosphorylation on the kinetic properties of ion channels. Moreover, the recent application of site directed mutagenesis to cloned ion channels has pinpointed specific amino acid residues critical for the specific kinase effects

Localisation of Putative Mechanoelectrical Transducer Channels in Cochlear Hair Cells by Immunoelectron Microscopy

Displacement of the apical stereociliary bundle of cochlear hair cells mechanically gates transducer channels. Knowing the position of the channels with regard to the apical structures of the hair cell could indicate how this mechanism operates. At present, there is conflicting evidence regarding their precise location; the channels have been suggested to be located either towards the base of the stereocilia or at the tips where they could be operated by extracellular links running from the top of shorter stereocilia to the sides of adjacent taller ones. The channels have been shown to be reversibly blocked by amiloride. This has prompted us to use a polyclonal antibody raised against another amiloride-sensitive channel to search for them using immunolabelling. The location of the primary antibody has been revealed using pre-embedding labelling with a colloidal gold-conjugated secondary antibody followed by scanning transmission electron microscopy of semi-thin sections. In this way, more complete information on the relationship of the labelling to the three-dimensional organisation of the stereociliary bundle has been obtained in comparison with previous immunofluorescence and transmission electron microscopic results. Labelling occurs in discrete areas towards the tips of the stereocilia, one of the possible sites for the transducer channels, predominantly between the membranes of shorter and taller stereocilia

Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Gαi-3 protein

We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha(i-3) subunit of the G(i-3) protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha(i-3) subunit was a member of this Na+ channel complex. G-alpha(i) structure and function were identified by Western blotting with specific G-alpha(i) subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G-alpha(i) proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha(i-3) subunit of the G(i-3) protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G-alpha(i-3) on Western blots of Golgi membranes which contain alpha(i-3) but not Na+ channel proteins, nor do they immunoprecipitate alpha(i-3) from solubilized Golgi membranes; however, alpha(i-3) is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha(i-3) and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha(i-3), but not alpha(i-2), stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha(i-3) protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation

Mapping the landscape: Peer review in computing education research

Peer review is a mainstay of academic publication – indeed, it is the peer-review process that provides much of the publications’ credibility. As the number of computing education conferences and the number of submissions increase, the need for reviewers grows. This report does not attempt to set standards for reviewing; rather, as a first step toward meeting the need for well qualified reviewers, it presents an overview of the ways peer review is used in various venues, both inside computing education and, for com- parison, in closely-related areas outside our field. It considers four key components of peer review in some depth: criteria, the review process, roles and responsibilities, and ethics and etiquette. To do so, it draws on relevant literature, guidance and forms associated with peer review, interviews with journal editors and conference chairs, and a limited survey of the computing education research community. In addition to providing an overview of practice, this report identifies a number of themes running through the discourse that have relevance for decision making about how best to conduct peer review for a given venue

CD133 Is a Marker of Bioenergetic Stress in Human Glioma

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho0 or ρ0, are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable up-regulation of CD133 through many cell passages. Moreover, these ρ0 cells display the ability to form “tumor spheroids” in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, ρ0 cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to ρ0 cells resulting in stable trans-mitochondrial “cybrid” clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised