Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the ␣2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits ␣, ␤1, and ␥2 and a number of GPCRs including M4, ␣2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and G␣ proteins shows specificity: there is interaction between the ␣2A receptor and Go, but little interaction between the ␣2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.T wo opposing ideas are invoked to explain how membrane bound signaling proteins transfer information after activation. In the first, components in the membrane freely diffuse and interactions occur through ''collision coupling'' determined by diffusion. Historically, such mechanisms are thought to govern the interaction of G protein-coupled receptor (GPCR) with G protein and the interaction of G protein with downstream enzymes and ion channels. Signal amplification is a key feature of this mechanism (1-3). A second mechanism is the ''physical scaffolding'' hypothesis in which component proteins interact directly or indirectly with each other. The best example of this is the role of InaD in the Drosophila photoreceptor that scaffolds via PDZ domains the light-sensing GPCR rhodopsin, Ca 2ϩ influx TRP channels, phospholipase C, and protein kinase C (4). In principle, this is a way of generating fast activation, fast signal termination, and specificity. A variant of this hypothesis is the localization of proteins in membrane signaling microdomains such as caveolae and lipid rafts.The G protein-gated K ϩ channel (GIRK) was first identified in atrial myocytes. Channel activation occurs after binding of acetylcholine to muscarinic M2 receptors (5) and is responsible for slowing of the heart rate in response to vagal stimulation (6, 7). Analogous GIRK currents are present in neurons and neuroendocrine cells (8). Activation of native and cloned GIRK channels has been shown to involve a direct, membrane-delimited interaction with the G␤␥ subunit (9, 10). One critical point is that the activation occurs rapidly in both native and heterologous settings: complete channel activation can occur within 1 s of the addition of agonist (11-13). Such fast rates of signaling suggest that the components diffuse only small distances, if at all. From these considerations alone it is an appealing hypothesis to propose that the components may be physically scaffolded together. O...

AimsAnecdotal observations suggest that sub-clinical electrophysiological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC) develop before detectable structural changes ensue on cardiac imaging. To test this hypothesis, we investigated a murine model with conditional cardiac genetic deletion of one desmoplakin allele (DSP ±) and compared the findings to patients with non-diagnostic features of ARVC who carried mutations in desmoplakin.Methods and resultsMurine: the DSP (±) mice underwent electrophysiological, echocardiographic, and immunohistochemical studies. They had normal echocardiograms but delayed conduction and inducible ventricular tachycardia associated with mislocalization and reduced intercalated disc expression of Cx43. Sodium current density and myocardial histology were normal at 2 months of age. Human: ten patients with heterozygous mutations in DSP without overt structural heart disease (DSP+) and 12 controls with supraventricular tachycardia were studied by high-density electrophysiological mapping of the right ventricle. Using a standard S1–S2 protocol, restitution curves of local conduction and repolarization parameters were constructed. Significantly greater mean increases in delay were identified particularly in the outflow tract vs. controls (P< 0.01) coupled with more uniform wavefront progression. The odds of a segment with a maximal activation–repolarization interval restitution slope >1 was 99% higher (95% CI: 13%; 351%, P= 0.017) in DSP+ vs. controls. Immunostaining revealed Cx43 mislocalization and variable Na channel distribution.ConclusionDesmoplakin disease causes connexin mislocalization in the mouse and man preceding any overt histological abnormalities resulting in significant alterations in conduction–repolarization kinetics prior to morphological changes detectable on conventional cardiac imaging. Haploinsufficiency of desmoplakin is sufficient to cause significant Cx43 mislocalization. Changes in sodium current density and histological abnormalities may contribute to a worsening phenotype or disease but are not necessary to generate an arrhythmogenic substrate. This has important implications for the earlier diagnosis of ARVC and risk stratification.

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.